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Biomedical subjects

J Eichberg

Publications and source records attributed to J Eichberg.

At least 19 recordsLinked to original sources
PubMed1994

Restricted hypotonic swelling of peripheral nerve myelin in streptozocin-induced diabetic rats.

Experimental diabetic neuropathy includes the non-enzymatic glycosylation (or glycation) of the major proteins of peripheral nerve myelin. We have used X-ray diffraction to determine whether such glycation affects myelin membrane structure and interactions in peripheral nerves from experimental diabetic rats. Streptozocin at 60 mg/kg was injected intraperitoneally to induce diabetes; controls were pair-fed and age-matched. Animals were sacrificed periodically from 2 weeks to after 1 year. The dissected sciatic nerves were tied off and incubated overnight at room temperature in hypotonic saline of defined pH and ionic strength or in distilled water. Such treatments have been shown to result in systematic changes of myelin period, which can be detected using X-ray diffraction, and which may indicate alterations in inter-membrane interactions owing to changes in composition. We observed no differences in repeat periods between control and diabetic nerves at pH 4.0 and 7.4, and ionic strength 0.01, 0.02, 0.06, 0.15, and 0.18; however, we did detect a significant difference (P < .02) in their maximum extent of swelling in distilled water: control nerves showed a period of 292 A (s.d. 23 A; n = 12) compared to 272 A (s.d. 19 A; n = 11) for diabetic nerves. To determine whether this difference in swelling was due to an alteration in the properties of the apposed, extracellular surfaces of the myelin membranes or to the connective tissue in peripheral nerve, we compared the X-ray patterns from peripheral nerve myelin isolated by sucrose density gradient centrifugation from sciatic nerves of diabetic and control rats. No difference in the patterns was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Guanosine-5'-(3-O-thio)triphosphate-mediated stimulation of phosphoinositidase C in solubilized rat peripheral nerve myelin and its alteration in streptozotocin-induced diabetes.

The regulation of phosphoinositidase C (PIC) activity by guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) was characterized in a cholate-solubilized peripheral myelin-enriched fraction from rat sciatic nerve. The GTP analog maximally enhanced PIC-catalyzed hydrolysis of exogenous phosphatidylinositol-4,5-bisphosphate (PIP2) in a dose-dependent manner only within a narrow range of cholate concentrations. Maximal stimulation was attained at 0.6 microM GTP gamma S and could be completely prevented by 1 microM guanosine-5'-(2-O-thio)diphosphate. Neither adenylyl-imidodiphosphate nor adenosine triphosphate (ATP) enhanced PIC activity. Carbamoylcholine (1 mM) added together with GTP gamma S increased the extent of PIP2 hydrolysis over that elicited by GTP gamma S alone and this stimulation was blocked by the muscarinic receptor antagonist, atropine (50 microM). In detergent-solubilized myelin preparations from streptozotocin-induced diabetic rats, a higher concentration of the guanine nucleotide analog was required to achieve stimulation comparable to that obtained with corresponding preparations from normal animals. These results suggest that sciatic nerve myelin possesses muscarinic receptors coupled via a GTP-binding protein to PIC and that this system can be reconstituted in detergent-solubilized extracts. It is possible that the function of G proteins in cell signaling is impaired in experimental diabetic neuropathy.

Adenosine Triphosphate↗
PubMed1994

P0 phosphorylation in nerves from normal and diabetic rats: role of protein kinase C and turnover of phosphate groups.

The effects of phorbol ester and forskolin on the net phosphorylation and turnover of P0 phosphate groups was studied in normal and experimentally diabetic rats. In sciatic nerve segments isolated from normal rats and incubated with [32P]-inorganic phosphate, phosphorylation of the major peripheral myelin protein, P0, was increased 2-5 fold in a time and dose-dependent manner by phorbol 12,13 dibutyrate (PDB). This increase was blocked by the protein kinase inhibitors, H-7 and staurosporine. Both the basal and PDB-stimulated phosphorylation of P0 were significantly greater in segments of sciatic nerve from streptozotocin-induced diabetic rats. Prolonged exposure of nerve segments to PDB abolished the stimulated phosphorylation of P0 and immunoblots of nerve proteins revealed a decrease in the content of the protein kinase C alpha-isoform. The adenylate cyclase activator, forskolin, had no effect on the PDB-stimulated phosphorylation of P0 in normal nerve but decreased phosphorylation in diabetic nerve. To measure turnover of P0 phosphate groups, nerves were incubated with 32P and incorporated label was then chased in radioactivity-free medium for up to 4 hours. P0 from normal nerve prelabeled under basal conditions lost 25% of its radioactivity during this time. In contrast, nearly all of the additional phosphate groups prelabeled in the presence of PDB disappeared after 2 hours of chase. P0 phosphate groups from diabetic nerve displayed similar turnover kinetics. When forskolin was added to the chase medium, the turnover of P0 phosphate moieties was accelerated in normal, but not in diabetic nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Decreased myo-inositol uptake is associated with reduced bradykinin-stimulated phosphatidylinositol synthesis and diacylglycerol content in cultured neuroblastoma cells exposed to L-fucose.

L-Fucose is a potent, competitive inhibitor of myo-inositol transport by cultured mammalian cells. Chronic exposure of neuroblastoma cells to L-fucose causes a concentration-dependent decrease in myo-inositol content, accumulation, and incorporation into phosphoinositides. In these studies, L-fucose supplementation of culture medium was used to assess the effect of decreased myo-inositol metabolism and content on bradykinin-stimulated phosphatidylinositol synthesis and diacylglycerol production. Chronic exposure of cells to 30 mM L-fucose caused a sustained decrease in bradykinin-stimulated, but not basal, 3H-inositol phosphate release and 32P incorporation into phosphatidylinositol in cells incubated in serum-free, unsupplemented medium. In addition, 32P incorporation into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was not altered in L-fucose-conditioned cells. Acute exposure of cells to serum-free medium containing 30 mM L-fucose did not affect either basal or bradykinin-stimulated 32P incorporation into phosphatidylinositol. Basal diacylglycerol content was decreased by 20% in cells chronically exposed to 30 mM L-fucose, although analysis of the molecular species profile revealed no compositional change. Bradykinin stimulated diacylglycerol production in neuroblastoma cells by increasing the hydrolysis of both phosphoinositides and phosphatidylcholine. Bradykinin-stimulated production of total diacylglycerol was similar for control and L-fucose-conditioned cells. However, there was a decrease in the bradykinin-induced generation of the 1-stearoyl-2-arachidonoyl diacylglycerol molecular species in the cells chronically exposed to 30 mM L-fucose. This molecular species accounts for about 70% of the composition of phosphoinositides, but only 10% of phosphatidylcholine. The results suggest that a decrease in myo-inositol uptake results in diminished agonist-induced phosphatidylinositol synthesis and phosphoinositide hydrolysis in cultured neuroblastoma cells grown in L-fucose-containing medium.

Animals↗
PubMed1994

Changes in Na-K ATPase and protein kinase C activities in peripheral nerve of acrylamide-treated rats.

In previous studies on rat peripheral nerve, we showed that acrylamide (ACR) exposure was associated with alterations in axonal and Schwann cell elemental composition that were consistent with decreased Na-K ATPase activity. In the present corollary study, the effects of ACR exposure on Na-K ATPase activity were determined in sciatic and tibial nerves. Subacute ACR treatment (50 mg/kg/d x 10 d, ip) significantly (p < .05) decreased Na-K ATPase activity by 45% in sciatic nerve but did not affect this activity in tibial nerve. Subchronic ACR treatment (2.8 mM in drinking water for 30 d) significantly decreased (p < .05) Na-K ATPase activities by 19% and 35% in sciatic and tibial nerves, respectively. Na-K ATPase activity was not altered in sciatic nerve homogenates exposed to 1.0 mM ACR in vitro. Since protein kinase C (PKC) has been proposed to play a role in the modulation of membrane Na-K ATPase function, PKC activity was also measured in sciatic nerve homogenates and subcellular fractions prepared from control and ACR-treated rats. Regardless of the ACR treatment protocol, PKC activity was elevated in nerve cytosol, but not in a particulate fraction. The results of this study suggest that decreased Na-K ATPase activity is involved in ACR-induced perturbation of axoplasmic and Schwann cell elemental composition in rat peripheral nerves and that loss of activity is not due to direct chemical inhibition of the enzyme. The role of PKC in ACR neurotoxicity requires further elucidation.

Acrylamide↗
PubMed1993

Effect of gangliosides on diacylglycerol content and molecular species in nerve from diabetic rats.

The effects of ganglioside treatment on 1,2-diacylglycerol content and on molecular species in 1,2-diacylglycerol, phosphatidic acid and total diacylglycerolipids, as well as Na+,K(+)-ATPase activity, were examined in sciatic nerves from streptozotocin-induced diabetic rats. Beginning 2 weeks after induction of diabetes, animals were administered mixed bovine brain gangliosides, AGF1, an inner ester derivative of this mixture, or saline for 5 weeks. The levels of 1,2-diacylglycerol and arachidonoyl-containing molecular species in age-matched non-diabetic animals were not affected by ganglioside treatment. In nerves from saline-treated diabetic animals, 1,2-diacylglycerol levels were not reduced, but both Na+,K(+)-ATPase activity and all arachidonyl-containing species except for 18:0/20:4 1,2-diacylglycerol were significantly decreased. The content of 1,2-diacylglycerol was lowered by 23 and 16% in bovine brain ganglioside and AGF1-treated diabetic animals, respectively, and the quantity of 18:0/20:4 1,2-diacylglycerol was also selectively reduced. Ganglioside administration did not affect the diminished levels of arachidonoyl-containing molecular species in 1,2-diacylglycerol, phosphatidic acid or diacylglycerolipids in nerve from diabetic rats. In the same nerves, bovine brain gangliosides partially and AGF1 completely restored Na+,K(+)-ATPase activity. The results suggest that gangliosides depress the content of total 1,2-diacylglycerol and the quantity of 18:0/20:4 1,2-diacylglycerol, specifically, in diabetic nerve. The possible relationship between the corrective action of gangliosides on Na+,K(+)-ATPase activity and the effect of these substances on 1,2-diacylglycerol molecular species composition and metabolism is discussed.

Animals↗
PubMed1993

Ganglioside treatment modifies abnormal elemental composition in peripheral nerve myelinated axons of experimentally diabetic rats.

Effects of ganglioside administration on elemental composition of peripheral nerve myelinated axons and Schwann cells were determined in streptozotocin-induced diabetic rats and nondiabetic controls. Diabetic rats (50 days after administration of streptozocin) exhibited a loss of axoplasmic K and Cl concentrations in sciatic nerve relative to control, whereas intraaxonal levels of these elements increased in tibial nerve. These regional changes in diabetic rat constitute a reversal of the decreasing proximodistal gradients for K and Cl concentrations that characterize normal peripheral nerve. Treatment of diabetic rats with a ganglioside mixture for 30 days (initiated 20 days after the administration of streptozocin) returned proximal sciatic nerve axoplasmic K and Cl concentrations to control levels, whereas in tibial axons, concentrations of these elements increased further relative to diabetic levels. Also in the ganglioside/diabetic group, mean axoplasmic Na concentrations were reduced and Ca levels were elevated. Mixed ganglioside treatment of nondiabetic rats significantly increased axoplasmic dry weight concentrations of K and Cl in proximal sciatic and tibial axons. Schwann cells did not exhibit consistent alterations in elemental content regardless of treatment group. Changes in elemental composition evoked by ganglioside treatment of diabetic rats might reflect the ability of these substances to stimulate Na+,K(+)-ATPase activity and might be related to the mechanism by which gangliosides improve functional deficits in experimental diabetic neuropathy.

Analysis of Variance↗
PubMed1993

Alterations in retinal Na+, K(+)-ATPase in diabetes: streptozotocin-induced and Zucker diabetic fatty rats.

The temporal pattern of changes in the specific activities of retinal Na+, K(+)-ATPase (Na, K-ATPase) and Mg(2+)-ATPase (Mg-ATPase) were determined at several time intervals following the onset of diabetes in streptozotocin-induced diabetic (STZ: at 1, 2, 4 and 6 months) Long-Evans hooded rats, spontaneously diabetic Zucker diabetic fatty (ZDF: at 1, 2 and 4 months) rats and their age-matched controls. These animals were utilized as models for insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM), respectively. Na, K-ATPase specific activity, using 10(3) M ouabain, was decreased (-6% to -14%) at all time points after the appearance of hyperglycemia in the ZDF rat, but was reduced only after 4 and 6 months in the STZ rat (-8% and -14%, respectively). In contrast, Mg-ATPase activity was significantly increased (13%) after 4 months in the ZDF rat and after 6 months in the STZ rat (8%). The concentration-dependent inhibitory effects of ouabain (10(-9) to 10(-3) M) on the activity of Na, K-ATPase in diabetic rats and age-matched controls was used to assess the time-dependent effects of diabetes on the alpha 3-high ouabain affinity or the alpha 1-low ouabain affinity retinal Na, K-ATPase isozymes. The retinal Na, K-ATPase activity for the alpha 3 isozyme was significantly lower at all times examined for the ZDF (-5% to -26%) and STZ-induced diabetic rats (-8% to -14%). This was reflected in the markedly decreased half-maximal inhibitory concentrations (IC50) of ouabain for the alpha 3 isozyme. For example, after four months of diabetes, the mean +/- SEM IC50 values were 12 +/- 3 nM in the STZ rats and 48 +/- 6 nM in the age-matched controls and 19 +/- 3 nM in the ZDF rats and 30 +/- 4 nM in the age-matched controls. In contrast, the activity of the alpha 1 isozyme was slightly, but significantly, decreased at 2 and 4 months in the ZDF rats (-4% to -7%) and after 4 and 6 months in the STZ-induced diabetic rats (-3% to -9%) while the IC50 values were unchanged. Moreover, the Hill coefficient for the alpha 3 isozyme was decreased in both diabetic groups while it was unchanged for the alpha 1 isozyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Activity and distribution of phosphoinositidase C in rat sciatic nerve.

The hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) by rat sciatic nerve cytosolic phosphoinositidase C [phosphoinositide-specific phospholipase C (PIC)] was studied at neutral pH and at ionic concentrations that approximate intracellular conditions. The principal water-soluble product formed was shown to be inositol trisphosphate by anion exchange chromatography. The maximum hydrolysis rate (2.5 nmol/min/mg protein) was achieved at less than 100 nM Ca2+. Hydrolysis was markedly increased to 15 nmol/min/mg protein by inclusion of K+ in the reaction mixture. In the presence of 200 mM K+, the optimum Ca2+ was increased to approximately 600 nM. Higher Ca2+ concentrations progressively inhibited PIP2 hydrolysis. Mg2+ also inhibited the reaction, but the presence of equimolar amounts of ATP and Mg2+ had no effect. Appreciable degradation of phosphatidylinositol-4-phosphate (PIP) also occurred in the nanomolar Ca2+ range, whereas breakdown of phosphatidylinositol (PI) required millimolar Ca2+. The presence of PIP but not PI inhibited PIP2 hydrolysis. Upon subcellular fractionation of nerve, more than 50% of recovered PIC activity was in the cytosol and about 20% was located in a myelin-enriched fraction. Using PIP2 as substrate, PIC activities in nerves from normal and streptozotocin-induced diabetic animals were not different. However, the myelin-associated enzyme from diabetic animals was more labile to freezing and thawing.

Animals↗
PubMed1992

Diacylglycerol composition and metabolism in peripheral nerve.

The content and molecular species composition of 1,2-diacylglycerol (DAG) in rat sciatic nerve was determined and compared with the molecular species profiles for glycerophospholipid classes in order to gain information concerning the metabolic pathways of DAG formation. The level of DAG in freshly dissected epineurium-free nerve (44 +/- 2 pmol/mg wet weight) was 10-40% of that in other tissues and cultured cells. The predominant DAG molecular species were 18:0/20:4 (30%) and 16:0/18:1 (17%). In comparison with phospholipid molecular species patterns, DAG was characterized by a substantial but lower proportion of the 18:0/20:4 species than was found in phosphoinositides, and a significant fraction of saturated species such as those found in phosphatidylcholine. In nerve from diabetic rats, both the content and arachidonoyl-containing molecular species of DAG were reduced. These species were also decreased in individual glycerophospholipids, except for phosphatidylinositol. The distribution of molecular species in phosphatidic acid (PA) did not resemble that of any other phospholipid. A large rise in DAG content occurred when nerve was incubated in vitro. Molecular species analysis indicated that phosphoinositides were the main source, especially during the initial period. This process was virtually abolished in a Ca(2+)-free medium and probably reflects a response to tissue injury. Evidence was obtained for the isomerization of DAG to 1,3-diacylglycerol during incubation. PA content and molecular species composition of incubated nerve did not change. However, inclusion of propranolol, a PA phosphatase inhibitor, caused a 40% accumulation of PA within 10 min, suggesting that formation of this phospholipid is continuous. These findings support the conclusion that DAG is principally derived from phosphoinositides by phospholipase C hydrolysis, but a minor fraction could be derived from phosphatidylcholine either by the action of phospholipase C or via phospholipase D and PA phosphatase. The metabolic origins of PA appear to be diverse.

Animals↗
PubMed1992

Decreased polyphosphoinositide metabolism accompanies myelinated fiber loss in human peripheral neuropathies.

The distribution of incorporated 32P in phospholipids of sural nerve biopsy samples from patients with several peripheral neuropathies was measured. Both the absolute amount and the proportion of isotope in polyphosphoinositides was decreased in nerves that displayed substantial (> 50%) depletion of myelinated fibers as compared to nerves that exhibited minimal depletion. The results suggest that diminished metabolism of these substances is an indicator of myelin loss, and are consistent with the conclusion that polyphosphoinositide turnover in human nerve is nearly entirely localized to the myelin sheath.

Adult↗
PubMed1992

Phorbol ester-mediated stimulation of phospholipase D activity in sciatic nerve from normal and diabetic rats.

Evidence for the presence of phospholipase D activity in sciatic nerve was obtained by incubation of 32P-prelabeled nerve segments in the presence of ethanol and measurement of [32P]phosphatidylethanol (PEth) formation expressed as a fraction of total phospholipid radioactivity. PEth synthesis was enhanced with increasing concentrations of ethanol (100 mM-2 M). 4-beta-Phorbol dibutyrate (100 nM-1 microM) stimulated PEth formation up to twofold in a time- and dose-dependent manner. The stimulatory effect evoked by 100 nM phorbol ester was completely abolished by Ro 31-8220 (compound 3), a selective protein kinase C inhibitor. Efforts to identify the phospholipid precursor of PEth were unsuccessful, suggesting this product arises from a small discrete precursor pool. On subcellular fractionation of nerve, the ratio of basal and 4-beta-phorbol dibutyrate-stimulated phospholipase D activity recovered in a myelin-enriched fraction, compared with a nonmyelin fraction, was 0.5 when results are expressed as a percentage of total phospholipid radioactivity. This ratio rises to 1.2 if the results are calculated assuming only phosphatidylcholine and phosphatidylethanolamine are potential precursors. The results suggest that myelin is a major locus of phospholipase D activity. Nerve from streptozotocin-induced diabetic and control animals displayed the same basal phospholipase D activity, but the enzyme in diabetic nerve was stimulated to a greater extent by a suboptimal concentration of 4-beta-phorbol dibutyrate. These results support the conclusion that protein kinase C modulates phospholipase D activity in nerve and suggest that in diabetic nerve the enzyme activation mechanism may possess increased sensitivity.

Animals↗
PubMed1992

Dopamine receptor-mediated activation of phospholipase C is associated with natriuresis during high salt intake.

Activation of phospholipase C (PLC) is considered to be one of the cellular signaling events involved in dopamine (DA)-mediated natriuresis. In the present study we have examined the role of renal cortical PLC in contributing to the increase in urinary sodium excretion during high sodium intake and its relationship with intrarenal DA synthesis. Rats were given either 1% NaCl (high sodium intake) or tap water (normal sodium intake) to drink for 24 h, and urine was collected over this time period. PLC activity in the renal cortex from these rats was measured by prelabeling cortical slices with myo-[2-3H]inositol and was expressed as fractional release (FR) of inositol (mono-, bis-, and tris-) phosphates. Acute increase in sodium intake produced 93 +/- 8% increase over control in urinary DA excretion. These changes were accompanied by significant increases (30 +/- 8%) in basal FR of inositol phosphates and 243 +/- 40 and 76 +/- 14% increases in urinary sodium and water excretion, respectively. The elevated basal PLC activity in rats with high sodium intake was significantly reduced in the presence of Sch 23390, a selective DA-1 receptor antagonist. Exogenously added DA (3 mM) also produced significant increases in PLC activity, although the magnitudes of increases were different in rats with high (37 +/- 8%) and normal (66 +/- 9%) sodium intake. However, Sch 23390 alone or carbidopa pretreatment did not affect the basal PLC activity in rats maintained on normal sodium intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1992

Diminished phospholipase C activation by dopamine in spontaneously hypertensive rats.

It is reported that a defect in dopamine-1 (DA-1) receptor adenylate cyclase coupling in the proximal convoluted tubule in the spontaneously hypertensive rat may contribute to the diminished natriuretic response to DA-1 receptor agonists. Since the tubular DA-1 receptor is also coupled to phospholipase C, and both of these cellular signaling processes are involved in DA-1 receptor-mediated diuresis and natriuresis, it is important to know whether a similar defect is also present in DA-1 receptor-coupled phospholipase C pathway. The present study was therefore designed to determine the functional status of DA-1 receptor-phospholipase C coupling system of adult spontaneously hypertensive rats using a renal cortical slice preparation. In addition, the renal response to exogenously administered dopamine (1 microgram/kg/min i.v.) was also determined. We found that basal phospholipase C activity was significantly higher in hypertensive rats than in age-matched Wistar-Kyoto rats (7.36 +/- 0.32% versus 5.61 +/- 0.27%, p less than 0.05). However, compared with the normotensive controls, dopamine-induced increases in phospholipase C activity were significantly attenuated in the preparations of hypertensive rats in a concentration-dependent manner (13 +/- 6% versus 38 +/- 6% for 1 mM dopamine, p less than 0.05; 49 +/- 6% versus 71 +/- 9% for 3 mM dopamine, p less than 0.05; 50 +/- 16% versus 106 +/- 22%, p less than 0.05 for 10 mM dopamine).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Characterization of receptors involved in dopamine-induced activation of phospholipase-C in rat renal cortex.

Dopamine (DA) is reported to stimulate phospholipase-C (PL-C) in rat renal cortex. Inasmuch as DA activates alpha adrenoceptors and DA receptors, the relative contribution of these receptors to DA-induced activation of PL-C is not yet established. We examined the effect of DA on PL-C activity in rat renal cortical slices prelabeled with myo-2-[3H]inositol in the presence of Li+. PL-C activity was expressed as fractional release (FR) of combined [3H]inositol phosphates expressed as dpm inositol phosphates accumulated/total dpm incorporated X 100. DA (1 mM) produced time-dependent increases in FR up to 60 min. DA (1, 3 and 10 mM) produced 61%, 88% and 110% increases in FR over control. When DA was given in the presence of SCH 23390, a selective DA-1 receptor antagonist, the increase in FR was significantly reduced to 33%, 51% and 62%, respectively, but the increase in FR remained unaffected in the presence of a DA-2 receptor antagonist, domperidone (30 microM). Phentolamine (10 microM) also inhibited the response to DA to 41%, 47% and 43% at the respective concentrations. DA-induced stimulation of PL-C was completely abolished in the combined presence of both SCH 23390 (30 microM) and phentolamine (10 microM). SCH 23390, domperidone or phentolamine alone did not significantly change the basal PL-C activity in renal cortical slices. These results demonstrate that 1) DA stimulates PL-C in rat renal cortex via activation of both DA-1 receptors and alpha adrenoceptors and DA-2 receptors are not involved in this response; and 2) during normal sodium intake, intrarenal DA does not modulate the PL-C activity in rat renal cortex.

Adrenergic alpha-Agonists↗
PubMed1991

Muscarinic cholinergic receptor-mediated phosphoinositide metabolism in peripheral nerve.

Few receptor-mediated phenomena have been detected in peripheral nerve. In this study, the ability of the muscarinic cholinergic receptor agonist carbamylcholine to enhance phosphoinositide (PPI) breakdown in sciatic nerve was investigated by measuring the accumulation of inositol phosphates. Rat sciatic nerve segments were prelabeled with myo-[3H]inositol and then incubated either with or without carbamylcholine in the presence of Li+. [3H]Inositol monophosphate ([3H]IP) accumulation contained most of the radioactivity in inositol phosphates, with [3H]inositol bisphosphate ([3H]IP2) and [3H]inositol trisphosphate ([3H]IP3) accounting for 7-8% and 1-2% of the total, respectively. In the presence of 100 microM carbamylcholine, [3H]IP accumulation increased by up to 150% after 60 min. The 50% effective concentration for the response was determined to be 20 microM carbamylcholine and stimulated IP generation was abolished by 1 microM atropine. Enhanced accumulation of IP2 and IP3 was also observed. Determination of the pA2 values for the muscarinic receptor antagonists atropine (8.9), pirenzepine (6.5), AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) (5.7), and 4-diphenylacetoxy-N-methylpiperidinemethiodide (4-DAMP) (8.6) strongly suggested that the M3 muscarinic receptor subtype was predominantly involved in mediating enhanced PPI degradation. Following treatment of nerve homogenates and myelin-rich fractions with pertussis toxin and [32P]NAD+, the presence of an ADP-ribosylated approximately 40-kDa protein could be demonstrated. The results indicate that peripheral nerve contains key elements of the molecular machinery needed for muscarinic receptor-mediated signal transduction via the phosphoinositide cycle.

Adenosine Diphosphate Ribose↗