 |
INTRODUCTION |
myo-Inositol (Ins)1 and
its polyphosphoinositide derivatives that are important in membrane
signaling have long been held to play a special role in brain
metabolism (1-4). However, the specific reason why Ins levels are
exceptionally high in mammalian brain tissue (4) is still unknown.
Although Ins is also an organic osmolyte (5-7), it would not appear
necessary for Ins to function in that capacity during normal
physiologic states, nor would an osmotic role easily explain the marked
microregional differences in Ins concentrations in brain (3). Based on
gas chromatography/mass spectrometry (GC/MS) analyses of minute brain
samples, there are apparent millimolar concentrations of Ins within
neurons (3), but hypertonicity-induced elevations in Ins in whole brain
tissue (6) may be largely confined to glial elements (7). As
polyphosphoinositides turn over rapidly and are also exceptionally
abundant in the nervous system (2, 4), millimolar levels of Ins may be
necessary to maintain differential rates of phosphoinositide synthesis. As an illustration, the depression in neuronal Ins levels and its
consequences on phosphoinositide turnover could be the biochemical basis for the effect of lithium on brain function (7-10). In contrast, the liver, an organ with diverse phosphoinositide species and high
rates of metabolic activities, has low micromolar levels of Ins, with
no evidence of an energy- and Na+-dependent,
high affinity transport system for Ins (11-13).
Synthesis of phosphatidylinositol (PtdIns), the most abundant
Ins-containing membrane phospholipid (14) by
phosphatidylinositol synthase
(CDP-diacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11), depends on adequate concentrations of the substrates, Ins
and CDP-diacylglycerol (15, 16). Its derivative,
phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2), is a
key molecule in the ubiquitous cell signaling system that utilizes
phospholipase C hydrolysis to yield the second messengers,
inositol-1,4,5,-trisphosphate (Ins-1,4,5,-P3) and diacylglycerol (17, 18). Such polyphosphoinositides, including the
products of PtdIns-3-kinase activities (19), and their inositol phosphate derivatives, such as inositol hexakisphosphate
(Ins-1,2,3,4,5,6-P6) (20, 21), also bind proteins at
specific domains (22-25). Protein-inositolpolyphosphate headgroup
interactions are important in membrane vesicle fission and fusion,
lipid rafts, the structure and function of the cytoskeleton, G
protein-gated ion channels, membrane transporters, and certain enzyme
activities (20-25). Rapid recycling of plasma membrane proteins may
depend on dynamic remodeling of membrane-bound inositol phosphate headgroups (26). In terms of phosphoinositide mass, it is likely that
protein-phosphoinositide binding, along with PtdIns-anchoring of
proteins to membrane bilayers (27), is the predominant occupation of
phosphoinositides within a cell, especially the neuron with its rich
cytoskeletal network and vesicle motor units.
The trapping of ~2-15 mM levels of Ins within a neuron
through active transport, restricted efflux, and relatively high
extracellular Ins levels as in cerebrospinal fluid may be
essential to its homeostasis (4). Concentration gradients of this
magnitude indicate a dependence on active Ins transport, especially at
the time of growth and differentiation (28). The
Na+/myo-Inositol cotransporter1
(SMIT1 or SLC5A3) is highly expressed prenatally
in central nervous system and placenta (5, 29-32). To gain more
insight into brain Ins metabolism, while ascertaining the importance of
SMIT1 as a transporter, we generated mice with a homozygous targeted
deletion of this gene and studied their phenotype.
 |
EXPERIMENTAL PROCEDURES |
Materials
The murine 129svJ genomic library was from Strategene (La Jolla,
CA). The restriction endonucleases were from Promega (Madison, WI). The
PGK-neomycin cassette was a gift from Dr. Nancy Cooke. The
HSV-tk-Dt
subunit cassette was a gift from Dr. Barbara Knowles.
The myo-inositol and
N,O-bis(trimethylsilyl)trifluoroacetamide
(BSTFA)/trimethylchlorosilane (TMCS) mixture were from Sigma. The
hexadeuterated myo-inositol
([2d6]Ins) was from CDN Isotopes
(Quebec, Canada). The Waymouth's MB 752/1 medium and
phosphate-buffered saline (PBS) were purchased from Invitrogen.
The paraformaldehyde and glutaraldehyde were from Electron Microscopy
Sciences (Ft. Washington, PA). The Triton X-100 was from Roche
Molecular Biochemicals. The [3H]acetate (100 mCi/mMol),
myo-[2-3H]inositol (22.3 Ci/mmol), and
[32P]dCTP (3000 Ci/mmol) were from PerkinElmer Life
Sciences. The halothane was from Abbott Laboratories, Inc. The sodium
borohydride, Superfrost glass slides and Tissue Freezing Medium were
from Fisher. All other chemicals were from Sigma.
Targeted SMIT1 Gene Deletion Construct
Previously, we cloned a genomic fragment that contained the
intron-free coding region of the mouse SMIT1 homologue (33) from a 129svJ genomic library. The fragment is ~15 kb and exists in a
bacteriophage vector. From this clone, an 11.0-kb
XbaI fragment was subcloned and used to construct a
targeting vector for homologous recombination by positive-negative
selection. A 1.6-kb EcoRI-NcoI fragment within
the SMIT1 coding region was replaced with a 1.9-kb
PGK-neomycin cassette (see Fig. 1). A HSV-tk-Dt
subunit cassette
was ligated to the 3'-end of the vector to allow negative selection of
the non-targeting events.
Homologous Recombination in Embryonic Stem Cells
Embryonic stem cells (129svJ) were electroporated, and
G418-resistant clones were isolated in the laboratories of
GenomeSystems, St. Louis, MO. In the laboratory of Dr. Gerard T. Berry,
a 1-kb SacI fragment 5'to the homology contained in the
targeting vector was used as a probe in Southern blot analyses to
identify homologous recombination events. HindIII digestion
of the wild-type locus generates a 6.5-kb fragment detectable by probe
1, whereas the correctly targeted locus yields a 10-kb fragment (see
Fig. 1). Twelve correctly targeted clones were identified among 192 resistant clones, yielding a targeting frequency of 1 in 16.
Generation of Chimeric Mice and Breeding of SMIT1(+/
) Mice
In the Transgenic Animal Facility of the University of
Pennsylvania, one embryonic stem clone was selected for blastocyst injection; of eight chimeric mice obtained, four transmitted embryonic stem cell DNA through the germ line and generated heterozygous offspring. Germline transmission was demonstrated by Southern blot
analysis of tail DNA.
SMIT1 Gene Expression in SMIT1(
/
) Mice
The content of SMIT1 transcript in adult, fetal, and placental
tissues was determined by Northern blot analyses.
myo-Inositol Analysis in Brain Tissue and the Whole
Embryo/Fetus
To maximize the sensitivity and specifically of Ins detection,
we employed GC/MS using hexadeuterated Ins
([2d6]Ins) in an isotope dilution
analysis. We employed trimethysilylation (TMS) and an
Hewlett-Packard 5890/5972 analyzer equipped with electron ionization in
the selected ion monitoring mode (3). The selected ion monitoring
fragments of interest are 217 for TMS-Ins and 220 for
TMS-[2d6]Ins. We generated a
linear standard curve using 1000 pmol of [2d6]Ins per derivatization vial,
with Ins varying from 200 to 1800 pmol. The final derivatization volume
containing BSTFA/TMCS with standards was 300 µl, and 1 µl was used
for each GC/MS injection. Based upon our results, this method
will permit picomolar amounts of Ins to be reliably assayed.
The isotope dilution analysis with
[2d6]Ins was used to measure Ins
levels in whole embryonic day (E) 10.5, E14.5, and E18.5 fetuses
from sets of litters and in amniotic fluid samples from one of the
E16.5 sets. The fetuses were quickly frozen in liquid nitrogen after
removal from the uterine sacs. The yolk sac DNA was used for
genotyping. A 5-µl sample of amniotic fluid was frozen and
lyophilized. The whole fetus was homogenized following addition of
[2d6]Ins. Aliquots were taken for
protein assay. Following lyophilization of fetal extracts, the
TMS-derivatized samples were analyzed by GC-MS.
Phospholipid (PL) and Surfactant Protein (SP) Analyses in Lung
Tissue and Electron Microscopy of Type 2 Pneumocytes
On day 18.5 of gestation, the uterus with fetuses intact was
removed and placed in sterile PBS on ice, and each fetal lung was
dissected out. Of the three lobes on the right side, one was fixed for
EM, one was fixed for immunostaining, and the third (largest) was
cultured with myo-[2-3H]inositol for labeling
of PtdIns. Of the two lobes on the left side, one was frozen
(
70 °C) for later use, and one (larger) was cultured for
[3H]acetate incorporation into all phospholipids. Lobes
were placed into individual wells of 24-well culture dishes with the
precursor (250 µl of Waymouth's media with either
[3H]acetate (20 µCi/ml, 1 mM total acetate)
or myo-[2-3H]inositol (10 µCi/ml, 7 µM Ins) and rocked for 5 h at three cycles/min in a
37 °C humidified incubator. Concentration of acetate was high (1 mM) to ensure rapid equilibration of the endogenous pools and eliminate pool effects. After incubation, the tissues were harvested into cold PBS, and then sonicated in saline and assayed for
protein. To determine distribution of incorporation into newly synthesized phospholipids, total lipid extracts were separated by
thin-layer chromatography (TLC), and the amount of tritium label in
each PL was determined as described previously (34).
To prepare frozen tissue sections for immunofluorescence, lobes were
fixed in 1% paraformaldehyde in PBS overnight at 4 °C, washed
with PBS for 5 min, and embedded in Tissue Freezing Medium (Fisher). Cut sections were washed (3 min) with sodium borohydride (0.1% in PBS) to reduce autofluorescence and rinsed twice with PBS (5 min). Sections were incubated (30 min, 25 °C) in PBS containing 0.3% Triton X-100 + 5% bovine serum albumin + 10% normal goat serum
to block nonspecific binding and permeabilize cells followed by a 5-min
wash with PBS + Triton X-100. Coverslips were incubated with primary
antibodies overnight at 4 °C. Antibodies used were: rabbit
anti-human SP-A (polyclonal), anti-bovine SP-B (polyclonal prepared
against SP-B extracted from bovine surfactant), and anti-rat SP-C
(polyclonal which recognizes precursor forms) as described previously
(35). To remove excess antibody, slides were incubated with PBS + Triton X-100 for 5 min.
Primary antibodies were detected by addition of secondary antibody
(goat anti-rabbit IgG conjugated to Cy3, 1:200) for 1 h at
25 °C. Excess secondary antibody was removed by 2-min washes (twice
each) with PBS + 0.3% Triton X-100, and then with PBS + 0.075% Triton
X-100, and finally with PBS. Coverslips were air-dried and mounted with
Mowiol (Calbiochem). Fluorescence was examined with an Olympus 1X70
microscope and Metamorph imaging system.
For electron microscopy, lobes were fixed in 2.5% glutaraldehyde, 0.1 M sodium cacodylate (pH 7.2) for 3 h at 4 °C,
washed, and postfixed with 1% osmium tetroxide. The tissue was
embedded in epoxy resin, and ultrathin sections were contrasted with
uranyl acetate and lead citrate and examined in a JEOL CX100II
transmission electron microscope operated at 80 kV. Cells from two
lungs of each group were examined (four sections/sample).
In Vitro Embryonic Mouse Preparation
The screening of respiratory motor pattern was double-blinded as
neither the genotype of the fetus was known at the time of this
recording nor were the results of the electrophysiological testing
known by the individual performing the Southern blot.
Brainstem/Spinal-diaphragm Preparations--
Fetuses
(E18.5) were delivered from timed-pregnant mice anesthetized with
halothane (1.25-1.5% delivered in 95% O2 and 5% CO2) and maintained at 37 °C by radiant heat. The timing
of pregnancies of dams was determined from the appearance of sperm
plugs in the breeding cages. Embryos were immediately decerebrated, and
the brainstem/spinal cord with the ribcage and diaphragm muscles
attached was dissected following procedures similar to those
established for perinatal rats (36, 37). The neuraxis was continuously perfused at 27 ± 1 °C (perfusion rate of 5 ml/min, chamber
volume of 1.5 ml) with Kreb's solution that contained: 128 mM NaCl, 3.0 mM KCl, 1.5 mM
CaCl2, 1.0 mM MgSO4, 24 mM NaHCO3, 0.5 mM
NaH2PO4, and 30 mM
D-glucose equilibrated with 95% O2, 5%
CO2 at 27 °C (pH = 7.4).
Medullary Slice Preparations--
Details of the preparation
have been described previously (38). Briefly, the brainstem/spinal
cords isolated from the E18.5 fetuses were pinned down, ventral surface
upward, on a paraffin-coated block. The block was mounted in the vise
of a vibratome bath (Leica, VT1000S). The brainstem was serially
sectioned in the transverse plane starting from the rostral medulla to
within ~100 mm of the rostral boundary of the pre-Bötzinger
complex (38), as judged by the appearance of the inferior olive. A
single transverse slice containing the pre-Bötzinger complex and
more caudal reticular formation regions was then cut (300-400 µm
thick), transferred to a recording chamber, and pinned down onto a
Sylgard elastomer. The medullary slice was continuously perfused in
physiological solution similar to that used for the brainstem/spinal
cord preparation except for the potassium concentration, which was
increased to 9 mM to stimulate the spontaneous rhythmic
respiratory motor discharge in the medullary slice.
Recording and Analysis--
Recordings of diaphragm
electromyography (see Fig. 4), hypoglossal (XII) cranial nerve root
(see Fig. 5), and population neuronal discharge within the
pre-Bötzinger complex (see Fig. 5) were made with suction
electrodes. Signals were amplified, rectified, low-passed filtered, and
recorded on computer using an analog-digital converter (Digidata 1200, Axon Instruments) and data acquisition software (Axoscope, Axon
Instruments). Mean values relative to control for the period and peak
integrated amplitude of respiratory motoneuron discharge were calculated.
 |
RESULTS |
Generation of SMIT1 Mutants--
Using a genomic clone containing
the entire coding region of the murine SMIT1 gene (33), we
prepared a targeted deletion construct of the murine SMIT1
gene (Fig. 1) and generated a homozygous deletion model. To obtain homozygous SMIT1 mutant (
/
)
mice, heterozygous F1 females and males were mated, and genotypes of their F2 offspring were analyzed at postnatal day 21. Of 170 F2 animals
collected, 63 of them were SMIT1(+/+), and 107 were
SMIT1(+/
). However, no SMIT1(
/
) mice were
detected. We subsequently determined that the SMIT1(
/
)
mice die shortly after birth. To determine the time of death of the
SMIT1(
/
) offspring of (+/
) × (+/
) matings,
cesarean sections were performed on E18.5 pregnant females. To confirm
that the fetuses were alive at the time of Cesarean section, each fetus
in the litter was stimulated by a gentle pinch with blunt end forceps
before removal of the uterus. All responded to this physical
stimulation by moving and extending extremities. By 20 min after the
Cesarean birth, (+/+) and the (+/
) fetuses had begun to breath, move,
squeal, and turn pink, whereas (
/
) pups became motionless and
cyanotic following an interval characterized by irregular gasps of
breath. All of the knock-out animals that were observed died within 20 min after birth.

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Fig. 1.
Diagram of the SMIT1
targeting vector and the wild-type and the mutant alleles.
PGK-neo, neomycin-resistant gene driven by
phosphoglycerokinase gene promoter; Dt , diphtheria toxin
-chain gene; ORF, open reading frame.
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|
Pathology of SMIT1(
/
) Fetus--
Body weight and
external features of the newborn (
/
) fetuses were
normal. In pathological examinations, there were no gross or
microscopic malformations. Routine hematoxylin/eosin-stained sections
of brain, spinal cord, dorsal root ganglia, heart, lungs, liver,
kidneys, esophagus, stomach, intestines, adrenal, thyroid glands, and
placenta appeared normal by light microscopy (data not shown).
SMIT1 Expression in SMIT1 Mutants--
The deletion of the bulk of
the SMIT1 coding region was confirmed by absent levels of
SMIT1 transcript, demonstrated by Northern blot analyses of total RNA
from fetal and placental samples (Fig. 2). In a survey of total RNA from
placenta and adult mouse tissues, the primary 11-kb SMIT1 transcript
was most abundant in kidney, placenta, and brain with weak expression
in thymus, lung, bladder, and testes (data not shown). In a survey of
poly(A) RNA-enriched samples from adult brain and kidney and placental
and embryo tissues, only an 11-kb transcript was detected (data not
shown). The SMIT1 11-kb transcript, demonstrated by Northern blot
analyses of total RNA from adult brain, was reduced in the heterozygous
(+/
) mice as compared with wild-type (+/+) (Fig. 2).

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Fig. 2.
SMIT1 expression in heterozygous
and homozygous mutant tissues. Each lane contained 30 µg of
total RNA. Hybridization was with 32P-labeled SMIT1
antisense riboprobe. +/+, wild-type; +/ , heterozygous; / ,
homozygous mutants. A, adult brain samples. Exposure time
was overnight. B, E14.5 placental samples. Exposure time was
overnight.
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|
myo-Inositol Quantitation in SMIT1 Mutants--
Using stable
isotope dilution gas chromatography/mass spectrometry with
hexadeuterated Ins ([2d6]Ins) in
an isotope dilution analysis, we measured the levels of Ins in fetal
brain, as well as whole embryos or fetuses obtained by cesarean
section. At E10.5, E14.5, and E18.5, the total body Ins content in
SMIT1(+/+) controls was 2.96 ± 0.45 (n = 8), 1.50 ± 0.13 (n = 3), and 1.16 ± 0.32 (n = 9) µmol/gram of wet weight, respectively (Fig.
3A). In age-matched
SMIT1(
/
) mutants, the levels were reduced by 77%
(n = 7), 64% (n = 6), and 84%
(n = 3), respectively, whereas in
SMIT1(+/
) heterozygotes, the levels were reduced by 52%
(n = 5), 32% (n = 6), and 43%
(n = 6), respectively. At E18.5, the Ins in isolated
brain tissue was 7.80 ± 0.80 (n = 7), 5.98 ± 1.50 (n = 5), and 0.60 ± 0.40 (n = 3) µmol/grams of wet weight from
SMIT1(+/+), (+/
), and (
/
), respectively (Fig.
3B). A similar trend was noted in amniotic fluid where the level of Ins was 480(n = 5), 325(n = 5), and 198 µM(n = 1) in the
SMIT1(+/+), (+/
), (
/
) samples, respectively. In
tissues obtained from control adult mice and analyzed by GC/MS, the
level of Ins in brain, kidney, and liver was 4.31 ± (n = 3), 4.26 ± 1.34 (n = 3) and
0.10 ± 0.03 µmol/grams of wet weight, respectively (mean ± S.D.). Thus, the whole SMIT1(
/
) near term fetus has an Ins level (
186 µM) comparable with those measured in
normal adult liver (
100 µM) and in a
SMIT1(
/
) amniotic fluid sample (198 µM).

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Fig. 3.
Whole body and brain Ins levels in
embryos/fetuses with SMIT1(+/+), (+/ ), and ( / )
genotypes. Using stable isotope dilution gas chromatography/mass
spectrometry with hexadeuterated Ins
([2d6]Ins) in an isotope dilution
analysis, the levels of Ins were measured in the whole embryo/fetus and
in brain tissue. A, at E10.5, E14.5, and E18.5, the whole
body Ins in SMIT1(+/+) controls, (+/ ) heterozygotes, and
( / ) homozygotes as micromoles/grams of wet weight. B, at
E18.5, the brain Ins levels in SMIT1(+/+) controls, (+/ )
heterozygotes, and ( / ) homozygotes as micromoles/grams of wet
weight.
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|
Surfactant Phospholipid Studies in Lung Tissue--
We considered
that the failure of SMIT1(
/
) pups to initiate effective
respiration could reflect abnormalities of pulmonary surfactant or type
II cell morphology. Therefore, precursor incorporation and PL analysis
in lung tissue was performed using E18.5 fetuses. There were no
differences in PL composition or synthesis detected by
[3H]acetate incorporation in the tissue from (
/
)
fetuses as compared with (+/+) or (+/
) fetuses (data not shown). The
acetate incorporation into total PL was 1.16 ± 0.09 (n = 7), 1.49 ± 0.15 (n = 11) and 1.34 ± 0.14 (n = 5) nmol/mg protein in lung
tissue from the SMIT1(+/+), (+/
), and (
/
) fetuses,
respectively. The relative myo-[2-3H]inositol
incorporation into PtdIns expressed as Ins/acetate was not lower in the
(
/
) lungs as compared with either wild-type (+/+) or (+/
) samples
(data not shown). The uptake of
myo-[2-3H]inositol into the aqueous fraction
of lung explants was also not different in samples from
SMIT1(
/
) fetuses as compared with (+/+) and (+/
)
fetuses (data not shown). Morphology of type II cells was examined by
both light and electron microscopy. By electron microscopy, the
ultrastructure of the type 2 pneumocytes in the (
/
) fetuses showed
no morphologic abnormalities, and the surfactant-containing lamellar
bodies appeared normal in both number and structure (data not shown).
By immunostaining, the relative density of type II cells appeared
similar in all groups, and the type II cells had normal contents and
subcellular distribution of the surfactant proteins, SP-A, SP-B, and
SP-C (data not shown).
Electrophysiological Analyses of Rhythmic Respiratory Neuronal
Activity--
In view of the normal findings related to pulmonary
surfactant in SMIT1(
/
) mice, it appeared likely that the
primary hypoventilation was due to nervous system dysfunction. To test
this hypothesis, recordings of inspiratory neuronal discharge were
obtained from multiple sites within the central neuraxis in E18.5
fetuses with different genotypes (Figs. 4
and 5). Diaphragm electromyography recordings from each of the
SMIT1(
/
) fetuses had very irregular respiratory rhythmic
patterns that had several characteristic features (Fig. 4). First, there were 3-8 apneic episodes of 15-60-s duration per a 10-min period. Second, the periods of suppressed respiratory rhythmic discharge were interspersed with bouts of rhythmic
respiratory bursting of much higher frequency than observed in
wild-type preparations. Third, there were regular occurrences of
augmented breaths that were of larger amplitude and slightly longer
duration. Fourth, the duration and amplitude of inspiratory bursts
(other than augmented bursts) in preparations from
SMIT1(
/
) fetuses were less than observed for the wild
type. Abnormal rhythmic discharge patterns were also observed in
recordings from the hypoglossal nerve rootlets of the medullary slice
preparations (Fig. 5). Direct recordings of brainstem neuronal
population discharge within the pre-Bötzinger complex (38)
demonstrated that the irregular rhythms were present in the putative
respiratory rhythm generating center, rather than simply reflecting a
failure of transmission of inspiratory drive to motoneuron populations
(Fig. 5). There were no significant differences in the respiratory
motor patterns generated in wild-type (+/+) versus carrier
(+/
) fetal in vitro preparations.

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Fig. 4.
Recordings of rectified and integrated
diaphragm electromyography (EMG) from in
vitro preparations isolated from E18.5 fetuses. The
drawing on the left depicts the brainstem/spinal
cord preparation with the diaphragm connected via the phrenic nerve. As
shown in A, the inspiratory rhythm was continuous and
regular in wild-type preparations. As shown in B, in
contrast, SMIT1( / ) preparations all had very irregular
respiratory rhythmic patterns that had several defining features. There
were 3-8 apneas of 15-60-s duration per 10-min period. Overall, the
interburst interval in SMIT1( / ) preparations (7.8 ± 8.1 s) was significantly longer than in SMIT1(+/+)
preparations (4.3 ± 2.4 s). As shown in C,
the periods of suppressed respiratory rhythmic discharge were
interspersed with bouts of rhythmic respiratory bursting of much higher
frequency (interburst interval of 1.4 ± 0.5 s) than observed
in wild-type preparations. Also, there were regular occurrences of
augmented breaths that were of larger amplitude and slightly longer
duration. As shown in D, the duration and amplitude of
rectified and integrated inspiratory bursts (other than augmented
bursts) in SMIT1( / ) preparations were ~65% of those
observed for (+/+) preparations.
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Fig. 5.
Recordings of integrated and rectified
inspiratory neuronal discharge in medullary slice preparations isolated
from E18.5 fetuses. As shown in A, the abnormal
rhythmic patterns were also observed in recordings from the hypoglossal
nerve rootlets of the medullary slice preparations from
SMIT1( / ) fetuses. PBC, pre-Bötzinger
complex. As shown in B, direct recordings of neuronal
population discharge within the PBC demonstrated that the irregular
rhythms were present in the putative respiratory rhythm generating
center. As shown in C, as was the case in the
brainstem/spinal cord preparation, the duration and amplitude of
rectified and integrated motor discharge (other than augmented bursts)
were decreased as compared with (+/+) preparation (recordings from XII
motoneurons).
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|
 |
DISCUSSION |
Our study shows that the SMIT1 transporter is the primary murine
transporter for the maintenance of an Ins concentration gradient during
embryonic and fetal life. This high affinity active transport system is
responsible for the Ins concentration gradient in the fetal-placental
unit. As shown in Fig. 3, a SMIT1 gene dosage effect is also
evident with heterozygotes having levels of Ins intermediate between
wild-type and knock-out fetuses. In the normal whole embryo and fetus,
the Ins levels continue to drop as the organism approaches term; the
levels are higher at E13.5 than at E14.5 or E18.5. This may be related
to de novo synthesis of Ins in the mammalian fetus and
placenta (14, 39-41) and reflected in the fetal plasma Ins
concentration that is significantly higher than the maternal level at
any time during gestation (39, 41-43). The plasma Ins level rapidly
declines after birth (39, 42), perhaps demonstrating the ability of
SMIT1 to concentrate, at the level of the placenta, the Ins produced by
both placental and fetal tissues. There is little movement of Ins from
mother to fetus or from fetus to mother (42, 43). The role of the low
affinity SMIT2 transporter in the maintenance of a cellular Ins
concentration gradient is unknown (44). It may be important during
fetal life when extracellular fluid concentrations are at their peak.
Based on the Ins concentrations in E18.5 fetuses and amniotic fluid,
our data are most compatible with a total collapse of the cellular Ins
concentration gradient at term gestation. This is the first model of
severe Ins deficiency in a mammal. The lethal hypoventilation can be
explained on the basis of central apnea and abnormal respiratory
rhythmogenesis generated within the pre-Bötzinger complex, the
putative respiratory rhythm generating center in the brainstem
(38).
One of the hypotheses that may explain the biochemical mechanism of
central nervous system dysfunction in the SMIT1(
/
)
newborn is that the marked reduction in Ins levels limits the activity of PtdIns synthase and as a consequence retards the production of
phosphoinositides. Potentially, this could lead to signaling abnormalities. Polyphosphoinositide binding to proteins might be
affected, resulting in an altered regulation of synaptic vesicle trafficking (26). In that regard, it is interesting that the synaptojanin-1 knock-out newborn pup with a selective deficiency of a
phosphatidylinositol-4,5-P2-5'-phosphatase isoform found in
clathrin-coated vesicles of nerve terminals has a phenotype that bears
many similarities to the SMIT1(
/
) newborn (26). This
genetic model suggests that a potentially lethal perturbation in
neurotransmission, as related to synaptic vesicle protein recycling, in
the first day of life is largely without effect in prenatal life.
Although a putative deficit in neuronal regulation in our model may be
present throughout the neuraxis because the phosphoinositide deficiency
is widespread, it is not surprising that the first recognizable
manifestation in postnatal life would involve the respiratory control
center pacemaker and present as abnormal respiratory rhythmogenesis.
Still unexplained, however, is the reason why Ins levels in murine
brain tissue are more than 6-fold higher (7.80 versus 1.16 µmol/grams of wet weight) than whole body levels at the time of
birth. This may indicate a special dependence of brain cells on Ins to
facilitate a process that, given our current knowledge of metabolic
pathways, is likely to involve phospholipids.
Based on measurements of tissue extracts or cultured cells derived from
such tissues, the apparent concentration of Ins in mammalian cells is
in both the micromolar and millimolar range. For example, millimolar
levels are found in the adult nervous system, kidney, and testes (1, 3,
14, 43), whereas micromolar levels have been detected in adult liver,
skeletal muscle, and heart (1, 12, 14, 43, 45). Micromolar
concentrations of Ins may be sufficient for de novo
synthesis of PtdIns as the Km of
phosphatidylinositol synthase for Ins has been reported to be in the
micromolar range when the assay was carried out using microsomes from
mammary gland and lung tissues (46, 47). Further support for the
concept that low micromolar concentrations of Ins could be sufficient
to allow for adequate PtdIns synthesis stems from work demonstrating
that low levels of Ins can redirect minor anionic phospholipid
synthesis in cultured type 2 pneumocytes (48). In a microsomal fraction
of lung tissue, as little as 4 µM Ins almost maximally
prevented the utilization of CDP-diacylglycerol for
phosphatidylglycerol synthesis (49). We found that an experimentally induced reduction in the estimated intracellular Ins concentration in
cultured fetal endothelial cells from ~5 to 1.5 mM did
not appear to lower the rate of PtdIns synthesis (50). Rate-limiting amounts of CDP-diacylglycerol in cellular membranes (51) may be more
important than variations in Ins concentrations between 2 and 15 mM (4) in the regulation of PtdIns synthesis. Our new
model, including the study of cultured cells derived from these mutant
animals, may provide information of a unique biological nature on the
adequacy of micromolar levels of Ins to fuel production of PtdIns in
diverse membranes within the neuron. If the levels of phosphoinositides
and their rates of synthesis in different brain regions of the SMIT1
knock-out mice prove to not be significantly reduced, then the
biochemical and physiological roles of Ins in the nervous system will
require further scrutiny.