Calcium Responses to Thyrotropin-Releasing Hormone, Gonadotropin-Releasing Hormone and Somatostatin in Phospholipase Cß3 Knockout Mice

Valerie A. Romoser, Thomas K. Graves, Dianqing Wu, Huiping Jiang and Patricia M. Hinkle

Department of Pharmacology and Physiology and the Cancer Center University of Rochester School of Medicine and Dentistry Rochester, New York 14642


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
These studies examined the importance of phospholipase Cß (PLCß) in the calcium responses of pituitary cells using PLCß3 knockout mice. Pituitary tissue from wild-type mice contained PLCß1 and PLCß3 but not PLCß2 or PLCß4. Both G{alpha}q/11 and Gß{gamma} can activate PLCß3, whereas only G{alpha}q/11 activates PLCß1 effectively. In knockout mice, PLCß3 was absent, PLCß1 was not up-regulated, and PLCß2 and PLCß4 were not expressed. Since somatostatin inhibited influx of extracellular calcium in pituitary cells from wild-type and PLCß3 knockout mice, the somatostatin signal pathway was intact. However, somatostatin failed to increase intracellular calcium in pituitary cells from either wild-type or knockout mice under a variety of conditions, indicating that it did not stimulate PLCß3. In contrast, somatostatin increased intracellular calcium in aortic smooth muscle cells from wild-type mice, although it evoked no calcium response in cells from PLCß3 knockout animals. These results show that somatostatin, like other Gi/Go-linked hormones, can stimulate a calcium transient by activating PLCß3 through Gß{gamma}, but this response does not normally occur in pituitary cells. The densities of Gi and Go, as well as the relative concentrations of PLCß1 and PLCß3, were similar in cells that responded to somatostatin with an increase in calcium and pituitary cells. Calcium responses to 1 nM and 1 µM TRH and GnRH were identical in pituitary cells from wild-type and PLCß3 knockout mice, as were responses to other Gq-linked agonists. These results show that in pituitary cells, PLCß1 is sufficient to transmit signals from Gq-coupled hormones, whereas PLCß3 is required for the calcium-mobilizing actions of somatostatin observed in smooth muscle cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Calcium ion serves as a key regulator of pituitary hormone secretion. Many calcium-mobilizing hormones act through G protein-coupled receptors to activate phospholipase Cß (PLCß), which catalyzes hydrolysis of phosphatidylinositol (4, 5) bisphosphate to inositol trisphosphate (IP3) and diacylglycerol. IP3 acts at IP3 receptors, calcium channels in the endoplasmic reticulum, to release intracellular calcium. There are four known PLCß isoforms, and it is apparent from both in vivo and in vitro studies that these exhibit selectivity with respect to their regulation. PLCß1 and PLCß3 are expressed in many tissues, whereas PLCß4 is limited to retina and certain neuronal tissues and PLCß2 to hematopoietic cells (1, 2). All PLCß enzymes can be activated by {alpha}-subunits of G proteins in the Gq family. PLCß1 and ß3 show high sensitivity to G{alpha}q (3, 4, 5, 6). PLCß2 and PLCß3 are also stimulated by G protein ß{gamma}-subunits (1, 7, 8, 9, 10, 11). In particular, ß{gamma}-subunits derived from the pertussis toxin-sensitive Gi and Go proteins have been implicated in activation of PLCß2 and PLCß3, accounting for the pertussis toxin-sensitive calcium transients sometimes seen when Gi/Go-linked receptors are activated, most notably in hematopoietic cells but also in neuronal, smooth muscle, and fibroblast cells. In vitro, G{alpha}q stimulates PLC- mediated phosphatidylinositol (4, 5) bisphosphate hydrolysis at much lower concentrations than Gß{gamma} (3, 11, 12).

In the anterior pituitary gland, TRH and GnRH are coupled to Gq and stimulate secretion, whereas somatostatin and dopamine are coupled to Gi/Go and inhibit secretion. TRH and GnRH produce strong calcium transients, initially by releasing intracellular calcium and subsequently by stimulating calcium influx (13, 14, 15). The role of different PLCß isoforms in these responses has not been clarified. The somatostatin and dopamine D2 receptors serve to inhibit release of hormones and reduce levels of hormone transcription in the pituitary gland, where they limit calcium influx (16, 17, 18, 19, 20). These various effects are achieved through either GTP-activated forms of G{alpha}i or G{alpha}o or through ß{gamma}-subunits from Gi/o proteins. Interestingly, somatostatin causes a calcium increase in certain nonpituitary tissues such as intestinal smooth muscle (21), apparently by activating PLCß3.

The balance of activating and inhibitory hypothalamic hormones is critical for normal pituitary function. It is not known which PLCß enzymes are expressed in pituitary, what role the different isoforms play, and why Gi/Go-coupled receptor systems produce different calcium responses in different cell types. We have taken advantage of mice with targeted disruption of the PLCß3 gene to determine the importance of this isoform in calcium responses to TRH and GnRH, which act on Gq-coupled receptors, and somatostatin, which acts via Gi/Go.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Role of PLCß3 in Calcium Signaling by Somatostatin
To examine the regulation of PLCß3 by receptors coupling via Gi/Go or the Gq family, we studied cells from PLCß3-deficient mice. PLCß3 null mice were generated by targeted gene disruption as described previously (22). At a gross phenotypic level, knockout animals (PLCß3-/-) were not significantly different from wild-type (PLCß3+/+) animals. The knockout mice were fertile and weights, growth rates, brain weight, behavior, and litter size were comparable in both sets of animals. There were no differences in plasma T4, corticosterone, glucose, lipids, or electrolytes in the two sets of mice, and no histological abnormalities were noted in pituitary, adrenal, thyroid, thymus, pancreas, testis, spleen, or liver from wild-type or PLCß3 knockout mice.

Somatostatin works through Gi/Go and in principle might stimulate PLCß3 by releasing Gß{gamma}. The peptide has been reported to mobilize intracellular calcium in smooth muscle cells (23, 24). We determined the ability of somatostatin to increase intracellular free calcium in aortic smooth muscle and anterior pituitary cells from PLCß3-knockout and wild-type animals. We also tested a mixture of agonists for Gq-linked receptors commonly expressed on cells (endothelin, bombesin, and bradykinin); these agonists (Gq mix) are expected to stimulate any PLCß by activating G{alpha}q. The combined Gq agonists were applied at the end of each experiment, and only those cells that responded to them with a calcium increase were considered potentially responsive to somatostatin, since these cells were viable with an IP3-releasable calcium pool.

Calcium Responses to Somatostatin in Aortic Smooth Muscle Cells from PLCß3+/+ and PLCß3-/- Mice
Cells prepared from the thoracic aorta of mice were stained with antibody against smooth muscle actin to verify cell phenotype (Fig. 1CGo). For measurement of intracellular free calcium, cells were loaded with fura2. The calcium responses of aortic smooth muscle cells from PLCß3+/+ and PLCß3-/- animals to agonists for Gq-coupled receptors were not significantly different, with 59 and 63% of cells responding with peak increases in 340/380 fluorescence ratios of 4.87 ± 0.05 and 5.03 ± 0.08, respectively (Fig. 1Go, A and D). In contrast, somatostatin caused an increase in calcium in aortic smooth muscle cells from wild-type but not knockout mice. In PLCß3+/+ mice, somatostatin increased calcium in 45% of responsive aortic smooth muscle cells, and the average peak height was 2.77± 0.08 times baseline (Fig. 1Go, B and D). Somatostatin mobilized calcium only at concentrations of 100 nM or greater. In contrast, no cells from PLCß3-/- mice (of 27 Gq-agonist responsive cells in 5 experiments) responded to somatostatin with any increase at all in intracellular calcium. These results indicate that the calcium response to somatostatin seen in smooth muscle cells requires PLCß3.



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Figure 1. Calcium Responses of Aortic Smooth Muscle Cells to G Protein-Coupled Receptor Agonists

Individual aortic smooth muscle cells isolated from PLCß3+/+ (A) or PLCß3 -/- (B) mice were treated with a mixture of Gq-linked receptor agonists (Gq mix: 50 nM bradykinin, 50 nM bombesin, 10 nM endothelin) or 1 µM somatostatin (SST) at the times indicated. Cell type was verified by immunofluorescent staining for smooth muscle-specific {alpha}-actin (C). The mean ± SE of the peak response in responsive cells, given as fold increase in 340:380 fluorescence ratio, is shown in panel D, and the percentage of cells responding is indicated in the bars.

 
Somatostatin also increased calcium in the fibroblast HEK293 cell line, which is in the same lineage as smooth muscle cells and expresses endogenous somatostatin receptors (Fig. 2AGo). The somatostatin response in HEK293 cells was observed in essentially all cells and was completely eliminated by pertussis toxin (Fig. 2BGo). All cells responded to Gq mix, but pertussis toxin had no effect on this response.



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Figure 2. Calcium Responses of HEK 293 Cells to G Protein-Coupled Receptor Agonists

HEK 293 cells were treated without (A) or with (B) 100 nM pertussis toxin (PTX) 18 h before performing calcium measurements. Cells were exposed to 1 µM somatostatin (SST) and Gq mix (50 nM bombesin, 50 nM bradykinin, 10 nM endothelin) at the times indicated.

 
Calcium Responses to Somatostatin in Pituitary Cells from PLCß3+/+ and PLCß3-/- Mice
Somatostatin did not increase intracellular calcium in anterior pituitary cells from wild-type and PLCß3-knockout mice. Because dopamine increases intracellular calcium at very low concentrations but not at high doses (25), we tested somatostatin over a range of doses from 1 nM and 10 µM, and monitored at least 30 cells at each concentration, but observed no calcium rise. In fact, we most often observed a slight decrease in intracellular calcium after somatostatin addition, presumably due to the inhibitory effect of the Gi/Go-linked somatostatin receptor on spontaneous voltage-gated calcium channels (data not shown). Somatostatin inhibits calcium influx in pituitary cells both by activating inward rectifier potassium current and by inhibiting L-type calcium channels (16, 18, 19). It seemed possible that a stimulatory effect mediated by PLCß3 might be masked by the effect of somatostatin to inhibit calcium influx. For this reason, we depolarized cells with high potassium, added nimodipine to inhibit L-type calcium channels, and then challenged cells with somatostatin (Fig. 3Go, A and B). Again, somatostatin never caused an increase in cytoplasmic calcium. To show that somatostatin receptors were functional, we added somatostatin after giving a mix of Gq-linked agonists (endothelin, bombesin, and bradykinin) or depolarizing with high potassium. In these paradigms, somatostatin decreased intracellular calcium in cells from both PLCß3+/+ and PLCß3-/- mice (Fig. 3Go, C and D). This indicates that the somatostatin signaling pathway, from receptor to G protein, was functioning effectively in pituitary cells from wild-type and knockout mice. The results also suggest that voltage-gated calcium entry accounts for the sustained calcium rise after activation of Gq-linked receptors.



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Figure 3. Calcium Responses of Pituitary Cells from PLCß3+/+ and PLCß3-/- Mice to Somatostatin

Cells from PLCß3+/+ (A and B) or PLCß3-/- (C and D) mice were exposed to 25 mM potassium chloride (KCl), 100 nM nimodipine (nimo), 1 µM somatostatin (SST), or a mixture of Gq-linked receptor agonists (Gq mix: 50 nM bradykinin, 50 nM bombesin, 10 nM endothelin) at the times indicated.

 
Both adenosine A1 and µ-opioid receptors, which are coupled to Gi/Go, have been demonstrated in pituitary cells from other species (26, 27), although they have not been characterized in mice. N6-cyclopentyladenosine and [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin (DAMGO) both increase intracellular calcium in smooth muscle cells (22, 23). We observed no increases in intracellular calcium when we treated pituitary cells from wild-type mice with the A1-selective adenosine receptor agonist N6-cyclopentyladenosine at 10 nM to 10 µM or the µ-opioid receptor agonist DAMGO at 100 nM to1 µM (data not shown). Dopamine, which was tested at 1 µM, also failed to increase calcium.

PLC Isoforms in PLCß3+/+ and PLCß3-/- Mice and HEK293 Cells
To determine which isoforms of PLCß are expressed in mouse pituitary cells, we performed Western blotting using pituitary tissue obtained from wild-type and PLCß3 knockout animals (Fig. 4Go). Samples from wild-type (+/+) and mutant (-/-) cell homogenates were loaded in equivalent amounts in each gel and blotted with antibodies against PLCß1, -2, -3, or -4. The specificity of antibodies to PLCß1, -2, and -3 was verified using purified PLCs and blocking peptides. In blots for PLCß4, we used cerebellar protein from wild-type and PLCß4 knockout animals (28) for positive and negative controls, respectively (Fig. 4DGo, lanes a and b); cerebellum is rich in PLCß4 (29). Anterior pituitary cells from either the wild-type or PLCß3-knockout mice expressed no detectable PLCß2 (Fig. 4BGo) or PLCß4 (Fig. 4DGo). As expected, PLCß3 was absent from pituitaries of knockout animals (Fig. 4CGo). There was, however, a significant amount of this isoform in the pituitaries of wild-type mice, as evidenced by the dark band. PLCß1 was present in pituitaries from PLCß3+/+ and PLCß3-/- mice (Fig. 4AGo). In three separate experiments, the intensity of the PLCß1 bands appeared the same for pituitary tissue from wild-type and knockout animals, indicating that PLCß1 is not generally up-regulated to compensate for the lack of PLCß3.



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Figure 4. Immunoblots for PLCß Isoforms in Mouse Pituitary

Homogenates from pituitaries of wild-type PLCß3+/+ (wt) and PLCß3-/- mice were resolved on SDS-PAGE and blotted with antisera against PLCß1 (A), PLCß2 (B), PLCß3 (C), or PLCß4 (D) at the following dilutions: PLCß1: 1:1000; PLCß2 and ß3: 1:500; PLCß4: 1:250. Samples from PLCß3+/+ (wt) and PLCß3-/- (-/-) were run in the indicated lanes. MW denotes lane with mol wt markers. Specificity of staining was verified by running samples of purified PLCß1, 2, and 3 and by absorbing antibodies with blocking peptides. Since neither purified PLCß4 nor blocking peptide was available, cerebellar homogenates from wild-type and PLCß4-/- knockout mice were run as a control; PLCß4 is abundant in cerebellum. Lanes a and b in panel D show cerebelli of wild-type or PLCß4-/- mice. Each lane was loaded with 0.4 µg of purified PLC or 40 µg of sample protein.

 
We asked whether smooth muscle and HEK293 cells express relatively more PLCß3 than pituitary cells, perhaps explaining why they respond to somatostatin with PLCß3 activation whereas pituitary cells do not. We were unable to use immunoblotting to estimate the relative concentrations of PLCß1 and PLCß3 in aortic smooth muscle cells because of the small numbers of cells available, but we were able to compare the relative amounts of different PLCß isoforms in HEK293 cells, which also show a calcium response to somatostatin (Fig. 2Go). As shown in Fig. 5Go, HEK293 cells express PLCß1 (panel A) and PLCß3 (panel B) but not PLCß2 (panel E). Immunoblotting was performed under the same conditions for pituitary and HEK293 cells. The ratio of PLCß3 to PLCß1 was similar in HEK293 cells, which respond to somatostatin with an increase in calcium, and pituitary cells, which do not.



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Figure 5. Immunoblots for PLCß Isoforms in HEK293 Cells

Proteins from HEK293 cells were resolved on SDS-PAGE and blotted with antisera against PLCß1 (A), PLCß2 (E), or PLCß3 (B) as described in the legend to Fig. 4Go. Lanes were loaded with 0.1 µg purified PLCß3 or 5 or 20 µl of a 20 µg/µl HEK293 cell supernatant or with 50 µg in the blot for PLCß2. Where noted, antisera against PLCß1 (C) or PLCß3 (D) were preabsorbed by incubating antisera at 100 µg/ml overnight at 4 C with 200 µg/ml specific blocking peptides before dilution and immunoblotting.

 
Gi/Go Proteins in Aortic Smooth Muscle and Pituitary Cells
We next asked whether differences in the levels of the cognate G proteins could explain the different calcium responses to somatostatin in different cell types. All known somatostatin receptors signal through G proteins in the Gi/Go family (17, 18, 20). We used immunoblotting to compare the relative concentrations of Gi and Go in homogenates from pituitary and aortic smooth muscle cells (Fig. 6Go). The amount of G{alpha}i staining was similar in both cell types. The antibody used recognizes isoforms 1, 2, and 3, which did not separate under the conditions of electrophoresis; the pattern shown in Fig. 6Go is typical for mouse G{alpha}i (30). The amount of G{alpha}o appeared to be somewhat lower in aortic smooth muscle than in pituitary cells. G{alpha}o is reported to run as three major bands representing the two splice variants of the protein and a posttranslationally modified form (31). These results suggest that the differences seen between pituitary and other cell lines are based on something more complex than the total G protein complement available.



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Figure 6. Immunoblots for G{alpha}

Homogenates from pituitaries (PIT) and aortic smooth muscle cells (ASMC) were resolved on SDS-PAGE and blotted with isoform-specific antibodies against G{alpha}o or G{alpha}i (common) at 1:250. Equivalent amounts of protein, 7.5 µg, were loaded in each lane.

 
Calcium Responses to GnRH and TRH in Pituitary Cells from PLCß3+/+ and PLCß3-/- Mice
The question of the relative contribution of each PLCß isoform to the calcium response to Gq-coupled receptors was addressed in pituitary cells of wild-type and PLCß3-deficient mice. TRH, GnRH, bombesin, bradykinin, and endothelin receptors all couple through G{alpha}q/11 to activate phosphatidylinositol turnover (15, 32, 33, 34, 35). Typical calcium responses of pituitary cells from PLCß3+/+ and PLCß3-/- mice to high doses of TRH, GnRH, or the Gq mix (50 nM bombesin, 50 nM bradykinin, 10 nM endothelin) are shown in Fig. 7Go. The responses of cells from intact and PLCß3 knockout mice appeared very similar. Upon agonist addition, there was a rapid increase in intracellular calcium due to release from intracellular stores, followed by a slow decline over a period of 50–200 sec. The proportion of cells responding and mean peak height attained after application of these agonists are shown in Fig. 8Go. Although the average responses to GnRH and TRH were comparable in wild-type and knockout pituitary cells, there was significant variability between individual cells within each experiment. Some cells responded robustly immediately, others exhibited a lag phase after agonist addition before responding, and some did not respond at all. We were not able to carry out an extensive analysis of the effect of sex and age on calcium responses, but in general, female pituitaries had slightly fewer TRH-responsive cells than their male counterparts (12–17% vs. 20%) (Table 1Go). Male and female pituitary cells gave comparable peak responses to 1 µM GnRH or TRH. There were no significant differences between mutant and wild-type pituitary cells in terms of percentages of responsive cells or the average maximal peak height for either sex (Table 1Go).



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Figure 7. Calcium Responses of Individual Pituitary Cells to Gq-Coupled Receptor Agonists

Cells from wild-type PLCß3+/+ (A–D) or PLCß3-/- mice (E–H) were treated with a mixture of Gq-linked receptor agonists (Gq mix: 50 nM bombesin, 50 nM bradykinin, 10 nM endothelin) (A and E), 1 µM TRH (B and F), 1 µM GnRH (C and G) or 1 nM GnRH (D and H) at the times indicated. Traces are representative of at least three experiments.

 


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Figure 8. Average Calcium Responses of Individual Pituitary Cells

Responses of pituitary cells from wild-type PLCß3+/+ (open bars) and PLCß3-/- (shaded bars) mice to two doses of TRH and GnRH were determined in experiments like that shown in Fig. 7Go. The mean ± SE of the peak response in responsive cells is shown, and the percentage of cells responding is indicated in the bars.

 

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Table 1. Summary of Calcium Responses of Pituitary Cells from PLCß3+/+ and PLCß3-/- Mice

 
To determine whether PLCß3+/+ and PLCß3-/- animals differed in their responses to low doses of Gq-coupled agonists, we tested GnRH and TRH at 1 nM, below the Kd values for the receptors (Fig. 7Go, D and H). The calcium mobilization patterns looked very similar to those resulting from higher concentrations of agonists or from the Gq cocktail. The lag time between addition of agonist and an increase in calcium was often longer at lower agonist doses, but the characteristic two-phase response was produced. Of note is that, again, the general shape of the responses was similar for wild-type and PLCß3-deficient cells. Between 35% (wild-type) and 38% (knockout) of cells responded to 1 nM GnRH. The percentage of responsive cells was somewhat higher with 1 nM than 1 µM GnRH (Fig. 8Go). This may be a reflection of the age and sex of the animals used, since lower-dose experiments were done with older males. In the case of 1 nM TRH, 16% (wild-type) to 21% (PLCß3-knockout) of cells responded with increases in intracellular calcium. When we reduced the concentration of GnRH and TRH to 0.1 nM, the fraction of responsive cells fell to approximately 5%, precluding quantitative analysis, but calcium responses were still observed in pituitary cells from the knockout mice.

Gq Staining in Pituitary Cells from PLCß3+/+ and PLCß3-/- Mice
Since G{alpha}q is expected to stimulate both PLCß1 and PLCß3, our results raise the question of how pituitary responses to Gq activation remained normal in PLCß3 knockout mice. As shown above in Fig. 4Go, PLCß1 was not up-regulated in pituitaries from knockout animals to compensate for the lack of PLCß3. Another possible means of compensation for the absence of PLCß3 is at the level of the G protein. Immunoblotting of protein samples from pituitaries for the common region of the {alpha}-subunits of Gq and G11 was unsuccessful because of the low abundance of this subunit. Immunofluorescent staining for Gq/11 {alpha}-subunit in pituitary cells from wild-type and mutant animals revealed no discernible difference in staining of cells from wild-type and knockout mice; both showed a largely membrane-bound distribution of G{alpha}q/11 (Fig. 9Go). The intensity of staining, indicative of the amount of protein present, was also comparable in both samples.



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Figure 9. Staining for G{alpha}q/11

Pituitary cells from PLCß3+/+ and PLCß3-/- mice were fixed and stained with antiserum against G{alpha}q C terminus, which recognizes both G{alpha}q and G{alpha}11. Staining was absent when primary antibody was omitted or when antibody was blocked with a G{alpha}q peptide before staining.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The somatostatin receptors are expressed in numerous neuronal and secretory tissues in the body, ranging from immune cells to kidney to endocrine tissues (17, 18, 36). All five isoforms of the somatostatin receptor (SSTR1–5, with splice variants SSTR2A and 2B) couple to Gi/o and are capable of decreasing the activity of adenylyl cyclase and increasing the activity of PLC when transfected into COS cells (17, 18, 20, 37). In the anterior pituitary, somatostatin plays an important role as a negative modulator of hormone secretion and gene transcription, inhibiting adenylyl cyclase and voltage-gated calcium channel activity and stimulating potassium channels and phosphatase activity (38, 39, 40). To our knowledge, somatostatin has never been shown to mobilize intracellular calcium in normal pituitary cells, but it has been shown to do so in the F4C1 pituitary cell line transfected with type 2 somatostatin receptor (41). Although the mechanisms involved in the diverse set of responses initiated by somatostatin receptor binding are not entirely defined, it is clear that the {alpha}- and ß{gamma}-subunits of Gi/o proteins, acting either individually or in concert, are central.

In smooth muscle cells, somatostatin can act as a calcium-mobilizing hormone (23, 27, 42). Somatostatin has been reported both to increase phosphoinositide turnover and intracellular calcium and stimulate contraction in various smooth muscle cell preparations (21, 24), although it has also been found to inhibit contraction (43).

Several lines of evidence indicate that in nonhematopoietic cells, Gß{gamma} stimulation of PLCß3 is responsible for IP3 production and calcium mobilization by Gi/Go-coupled receptors, including somatostatin receptors. For example, overexpression of transducin {alpha}, which binds Gß{gamma}, inhibits calcium responses via pertussis toxin-sensitive receptors (9), as does antibody to PLCß3 and Gß{gamma} (23). Our data showing that somatostatin does not increase calcium in cells from PLCß3-knockout mice provide additional strong support for this model. Together with the finding that calcium responses via the µ-opioid receptor are absent in the PLCß3-/- mice (22), these results can be extrapolated to suggest that pertussis toxin-sensitive calcium responses in nonhematopoietic cells in general are caused by Gß{gamma} activation of PLCß3.

It is less clear why somatostatin fails to initiate a calcium transient in cell types like pituitary, where somatostatin evokes other calcium responses. Differences in receptor subtypes or density, G protein repertoire, or effector abundance all have the potential to affect the coupling of the receptor. Mouse pituitary and aortic smooth muscle cells were not attainable in sufficient quantities to permit analysis of somatostatin receptor subtype expression, but all somatostatin receptor subtypes are expressed in pituitary glands of other species and the subtypes most effective in activating PLCß3, SSTR2 and 5, are present at high levels in somatotrophs (21, 39, 40, 44). Pituitary cells from wild-type and PLCß3-/- mice showed normal inhibitory responses to somatostatin, indicating that they had an intact somatostatin receptor-G protein pathway. We also showed that pituitary cells express the G protein partners of the somatostatin receptor, Gi and Go, at densities at least equivalent to those in aortic smooth muscle cells, although we did not rule out possible differences in G{alpha}i subunits. Finally, we showed that the requisite effector is present, because wild-type mouse pituitary contained PLCß3 in quantities equivalent to those in responsive fibroblasts, although the distribution in different pituitary cell types is not known. One potential explanation for the failure of pituitary cells to show a calcium response to somatostatin is that alternative effectors such as calcium channels engage available Gß{gamma}. Another is that pituitary somatostatin receptors are spatially organized in a manner that favors regulation of ion channels and adenylyl cyclase but minimizes activation of PLCß3. Since some somatostatin receptor subtypes have PDZ domain-binding motifs at their carboxyl termini, they may interact with scaffolding proteins that could be expressed in a cell type-specific pattern. A number of proteins with PDZ domains have recently been shown to interact with the type 2 somatostatin receptor (45, 46). It is not obvious how this would account for activation of PLCß3 in some cells but not others, because PLCß3, according to fluorescent microscopy, appears to be distributed throughout the cytoplasm (data not shown). Furthermore, calcium mobilization through the type 2 receptor reportedly does not require the C terminus of the receptor (41).

TRH and GnRH both exert their primary effects via Gq/11-mediated activation of PLCß. To our knowledge, single cell responses of mouse pituitary cells have not been described previously. Not surprisingly, the characteristics of the responses were quite similar to those measured in rat pituitary glands under very similar conditions (47). Our findings that TRH and GnRH responses are unaffected by disruption of the PLCß3 gene imply that G protein activation of PLCß1 is sufficient to support a full calcium response. The fact that PLCß3 knockout mice develop normally, are normal size, and are fertile is consistent with our finding of normal TRH and GnRH responsivity. The only phenotype known to result from disruption of the PLCß3 gene is exaggerated sensitivity to morphine analgesia (22) and formation of skin ulcers on the neck and behind the ears (48). It appears that the ability of Gi/Go-linked receptors to increase calcium in smooth muscle is nonessential. We have shown that the pituitary gland normally expresses just two G-protein- regulated PLCs, ß1 and ß3. Mice with targeted disruption of the PLCß1 gene have been described and are reported to live for only a few weeks (49). Their growth is severely retarded, consistent with a critical role of PLCß1 in endocrine function. The mice also suffer from severe seizure disorders.

A major question raised by our data is why elimination of PLCß3, which is strongly activated by the {alpha}-subunit of Gq/11, had no effect on calcium responses to Gq-activating hormones. We considered the possibility that high doses of TRH and GnRH increased IP3 to levels above those necessary for a maximal calcium response, masking possible differences. However, we were unable to detect differences between wild-type and knockout animals when we tested hormones at concentrations so low that only rare cells responded. We ruled out the possibility that PLCß1 was up-regulated in pituitary as a whole, although we did not determine its distribution in individual cells, and we ruled out the possibility that a PLCß isoform not normally expressed in pituitary was turned on in the PLCß3-/- mice. Although immunocytochemistry is not quantitative, we also saw no difference in staining for the common region of the {alpha}- subunits of Gq and G11, the relevant G proteins. Taken together, these findings suggest that cells do not compensate for the lack of PLCß3 by increasing the concentration of receptors, G proteins, or PLCß1. There may be another mechanism for compensation for the lack of PLCß3. Alternatively, TRH and GnRH may normally stimulate PLCß1 but not ß3; if so, the basis for such specificity is unknown. The activity of PLCß3 can be inhibited by a cAMP-dependent phosphorylation (50), and it could be that the levels of cAMP in the pituitary are sufficient to render PLCß3 largely inactive under normal conditions. The PLC enzymes may be functionally compartmentalized, or there may be accessory proteins that promote Gq activation of PLCß1 or prevent activation of PLCß3.

Our studies with PLCß3 knockout mice provide compelling evidence that stimulation of PLCß3 by Gß{gamma} leads to the release of intracellular calcium in those tissues where activation Gi/Go-coupled receptors provoke an increase in calcium. PLCß3 is abundant in anterior pituitary, even though agonists for Gi/Go- coupled receptors do not release intracellular calcium in intact pituitary cells. Calcium responses to TRH and GnRH appeared to be intact in pituitaries from PLCß3-/- mice, showing that PLCß1 is adequate for normal signal transduction.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Cell culture media, sera, HBSS, antibiotics, collagenase, and trypsin were from Life Technologies, Inc. (Gaithersburg, MD). Cell-Tak was from Collaborative Biomedical Products (Bedford, MA). Plasticware for cell culture was from Falcon (Lincoln Park, NJ). Fluorescent indicators were from Molecular Probes, Inc. (Eugene, OR). TRH was from Calbiochem (La Jolla, CA) and antiserum to smooth muscle actin, other hormones, and protease inhibitors were from Sigma (St. Louis, MO). Affinity-purified PLCß2 and ß3 antibodies were kindly provided by Dr. Alan Smrcka (University of Rochester, Rochester, NY), who also donated purified PLCß3. Additional antibodies against PLCß1 and PLCß3, antibodies against PLCß4 and G protein {alpha}i and {alpha}o subunits, and blocking peptides were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Purified antiserum to G{alpha}q/11 was a generous gift from Dr. Paul Sternweis (University of Texas, Dallas, TX). Rhodamine- and fluorescein-conjugated secondary antibodies were from American Qualex (LaMiranda, CA).

Calcium Measurement
Measurements of intracellular calcium were performed at 37 C essentially as described by Nelson and Hinkle (51). Cells were loaded at room temperature with 4 µM Fura2-AM, 0.2% BSA, and 20 µg/ml cyclosporin A for 40–50 min in the dark in HBSS buffered with 15 mM HEPES to pH 7.4, rinsed, and placed in a Sykes-Moore chamber from Bellco (Vineland, NJ) covered with 1 ml HBSS. Single-cell calcium experiments and imaging were performed on a Nikon inverted microscope with a DAGE CCD72 camera and Geniisys intensifier system (Michigan City, IN) using Image-1 or Metafluor software from Universal Imaging Corp. (Media, PA). Results show traces from individual cells within a field. All traces depict cells that were responsive to a mixture of Gq-activating agonists, and all experiments were repeated at least three times with comparable results.

Pituitary and aortic smooth muscle cells were obtained from 6- to 12-week-old mice after cervical dislocation or CO2 asphyxiation. Between two and four animals were used for each preparation. Pituitary glands were transferred under aseptic conditions to 100 µl of 1.25 mg/ml trypsin in EDTA and chopped finely with a razor blade. The tissue was then enzymatically dispersed by two 5-min and one 10-min incubation in 1 ml trypsin at 37 C, followed by one 20-min incubation in 1 mg/ml type I collagenase in DMEM. Cells were dispersed mechanically with a pipette tip, resuspended in growth medium [DMEM supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), fungizone (1 µg/ml), and kanamycin (100 µM)], and plated on Cell-Tak-coated glass coverslips in growth medium and grown 24–48 h before use. For calcium experiments, cloning rings were used to confine the cells in a small area.

To isolate aortic smooth muscle cells, a 1-cm length of thoracic aortic tissue was removed and rinsed briefly in sterile HBSS. The tissue was then transferred to 1 ml of sterile HBSS containing 1.3 mg/ml type I collagenase and 0.3 U/ml type I elastase and incubated 40 min at 37 C. Adventitia were removed and the tissue placed in 1 ml sterile HBSS with 2 mg/ml collagenase and 3.3 U/ml elastase and minced using a sterile razor blade. After a 60-min incubation at 37 C, a final enzymatic digestion in 1 ml of 3 mg/ml collagenase for 20 min at 37 C was performed. Cells and remaining tissue fragments were mechanically dispersed, and the cells were collected by centrifugation, resuspended in growth medium, and plated on coverslips coated with poly-L-lysine. Cells were grown at 37 C in a humidified atmosphere and the medium was changed daily until they had reached near confluence, within 4–7 days. Cell type was verified by immunofluorescent staining for smooth muscle-specific actin.

Protein Isolation and Western Blotting
For immunoblotting, aortic smooth muscle cells were isolated as above and grown in 35-mm dishes. Cells were washed and collected in HBSS, and then spun at 3,000 rpm. The cell pellet was resuspended in lysis buffer (25 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 µg/ml aprotinin, 0.2 µg/ml leupeptin, 1 µg/ml pepstatin A, 42 µg/ml tosylargininemethylester, 21 µg/ml tosylphenylalaninechloroketone, 133 µM phenylmethanesulfonylfluoride) and the cells were disrupted by four cycles of freeze thawing. After addition of NaCl to make the solution 1 M NaCl, the cell slurry was spun at 60,000 rpm in a Beckman Coulter, Inc. ultracentrifuge 20 min at 4 C. The supernatant fraction containing cytosolic proteins was stored at –70 C until use. The pellet was resuspended in lysis buffer containing 100 mM NaCl and 1% cholate and extracted on ice for 30 min. After another centrifugation at 60,000 rpm at 4 C, the supernatant fraction containing membrane proteins was frozen at –70 C until use.

For immunoblotting, pituitaries were collected from 10–40 animals and rapidly frozen in DMEM with 10% FBS and 10% dimethylsulfoxide in liquid nitrogen, then stored at –70 C until use. Pituitaries were then thawed and minced finely with a sterile razor blade in a small volume of homogenization buffer (10 mM Tris, pH 7.4, 5 mM EDTA, 1 mM dithiothreitol, and protease inhibitors as above) and homogenized in a Dounce homogenizer at 4 C. The slurry was spun at 17,000 x g for 15 min at 4 C, and the pellet was resuspended in two volumes of homogenization buffer containing 1% cholate. After 1 h at 4 C with occasional mixing, the tube was centrifuged as above and the supernatant fraction was used for immunoblotting. SDS polyacrylamide gels (9 or 12% polyacrylamide) were run and proteins were electrophoretically transferred to nitrocellulose membranes. For Western blotting, primary antibodies were used at the concentrations indicated; horseradish peroxidase-conjugated secondary antibody was used at 1:2,000–1:5,000 dilution and proteins were visualized with enhanced chemiluminescence.

Immunofluorescence Microscopy
Immunocytochemistry was performed essentially as described (52). All steps were carried out at room temperature. Cells grown 24–48 h on glass coverslips were treated as indicated in experiments, rinsed three times in PBS, and fixed in a 4% paraformaldehyde solution in PBS for 30 min. After three washes with PBS, cells were permeabilized in blocking buffer containing 0.2% Nonidet P-40 and 5% goat serum in serum-free Ham’s F10 medium. Coverslips were incubated for 60–180 min in primary antibody diluted as indicated in blocking buffer. Cells were washed four times for 5 min with PBS and then incubated for 30 min with fluorescently tagged secondary antibody at 1:100 dilution in blocking buffer. Images were captured using Metamorph software.


    ACKNOWLEDGMENTS
 
We are grateful to John Puskas for excellent technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Dr. Patricia M. Hinkle, Department of Pharmacology and Physiology, Box 711, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, New York 14642. E-mail: Patricia_Hinkle{at}urmc rochester.edu.

This work was supported by NIH Grant DK-19974 (P.M.H.) and Mentored Clinical Scientist Award DK-02439 (T.K.G.).

Received for publication August 7, 2000. Revision received October 6, 2000. Accepted for publication October 13, 2000.


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