Insulin stimulates membrane conductance in a liver cell line: evidence for insertion of ion channels through a phosphoinositide 3-kinase-dependent mechanism.

Activation of insulin receptors stimulates a rapid increase in the ion permeability of liver cells. To evaluate whether this response involves insertion of ion channels, plasma membrane turnover was measured in a model liver cell line using the fluorescent membrane marker FM1-43. Under basal conditions, the rate of constitutive membrane turnover was approximately 2%min(-1), and balanced exocytosis and endocytosis maintained the total cell membrane area constant. Exposure to insulin stimulated a transient increase in membrane turnover of up to 10-fold above constitutive rates. The response was concentration-dependent (0.001-10 microm). Insulin also caused a parallel increase in membrane conductance as measured by whole-cell patch clamp recording due to opening of Cl(-)- and K(+)-selective ion channels. The insulin-stimulated membrane turnover did not appear to involve the constitutive recycling compartments, suggesting that a distinct pool of vesicles may be involved. The effects of insulin on membrane turnover and membrane conductance were inhibited by blockers of phosphoinositide 3-kinase LY294002 and wortmannin or by disrupting microtubule assembly with nocodazole. Taken together, these findings indicate that insulin stimulates recruitment of new membranes through phosphoinositide 3-kinase-dependent mechanisms. Thus, regulated insertion of a separate population of ion channel-containing vesicles may represent one mechanism for mediating the changes in membrane conductance that are essential for the cellular response to insulin.

ClC-2 chloride channels contribute to HTC cell volume homeostasis.

Membrane Cl(-) channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl(-) currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl(-) currents when non-ClC-2 currents were blocked by DIDS (100 microM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl(-) currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl(-) permeability and cell volume homeostasis.

Ezrin-radixin-moesin-binding phosphoprotein 50 is expressed at the apical membrane of rat liver epithelia.

Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) and NHE3 Kinase A regulatory protein (E3KARP) are membrane-cytoskeleton linking proteins that utilize 2 PSD-95/DIg/ZO-1 (PDZ) domains and an ERM binding site to coordinate cyclic adenosine monophosphate (cAMP)-regulated ion transport in a number of distinct epithelia. ERM family members serve to anchor EBP50 and E3KARP to the actin cytoskeleton and sequester protein kinase A (PKA) to these protein complexes. In hepatocytes and cholangiocytes, the epithelial cells of the bile secretory unit, cAMP-activated PKA stimulates secretion and bile formation, but the molecular mechanisms, including the potential contribution of EBP50 and E3KARP, remain undetermined. The present studies evaluated the comparative expression and localization of EBP50 and E3KARP in rat hepatocytes and cholangiocytes. Complementary DNAs encoding rat EBP50 and E3KARP were identified by reverse transcription-polymerase chain reaction in both epithelial cell types and subsequently sequenced. Northern and Western analysis showed the presence of EBP50 messenger RNA and protein in both hepatocytes and cholangiocytes. Confocal immunofluorescence revealed EBP50 was concentrated at the apical domain of both cell types. E3KARP was also expressed in cholangiocytes but had a distinct cytoplasmic/nuclear distribution. In dominant-negative transfection studies, patch clamp analysis of Mz-ChA1 cholangiocarcinoma cells showed that expression of the PDZ1 domain of EBP50 selectively decreased the endogenous cAMP-mediated Cl secretory response. The apical expression of EBP50, presence of specific ERM proteins, and functional effects of PDZ1 expression on cholangiocyte secretion suggest EBP50 is positioned to contribute to the organization and regulation of bile secretory proteins in both hepatocytes and cholangiocytes.

Evidence for ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) self-association through PDZ-PDZ interactions.

Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a versatile membrane-cytoskeleton linking protein that binds to the COOH-tail of specific integral membrane proteins through its two PDZ domains. These EBP50 binding interactions have been implicated in sequestering interactive sets of proteins into common microdomains, regulating the activity of interacting proteins, and modulating membrane protein trafficking. With only two PDZ domains, it is unclear how EBP50 forms multiprotein complexes. Other PDZ proteins increase their breadth and diversity of protein interactions through oligomerization. Hypothesizing that EBP50 self-associates to amplify its functional capacity, far-Western blotting of cholangiocyte epithelial cell proteins with EBP50 fusion protein revealed that EBP50 binds to a 50-kDa protein. Far-Western blotting of EBP50 isolated by two-dimensional gel electrophoresis or immunoprecipitation demonstrates that the 50-kDa binding partner is itself EBP50. Further, co-transfection/co-precipitation studies show the self-association can occur in an intracellular environment. In vitro analysis of the EBP50-EBP50 binding interaction indicates it is both saturable and of relatively high affinity. Analysis of truncated EBP50 proteins indicates EBP50 self-association is mediated through its PDZ domains. The ability to self-associate provides a mechanism for EBP50 to expand its capacity to form multiprotein complexes and regulate membrane transport events.

Coincident microvillar actin bundle disruption and perinuclear actin sequestration in anoxic proximal tubule.

The present studies investigated acute disruption of microvillar actin cytoskeleton and actin association with other cytoskeletal components in ATP-depleted rabbit proximal tubular cells. Video-enhanced differential-interference contrast microscopy and confocal microscopy were used to follow the fate of F-actin during the disruption of microvilli. Within individual cells, all microvilli collapsed simultaneously. Microvillar actin filaments underwent a parallel decrease in length. Using a sequential cytoskeletal extraction protocol and electron microscopy, we revealed in the present studies the coincident sequestration of a distinct, perinuclear pool of actin that was primarily absent in control cells. Actin sequestration progressed in a duration-dependent manner, occurring as early as 15 min of anoxia when cellular ATP dropped to <5% of control level. Phalloidin staining and depolymerization treatment showed the majority (>90%) of this sequestered actin to be F-actin. A microvillar actin bundling protein villin was also sequestered in the same perinuclear complex of anoxic proximal tubules. In conclusion, the present results demonstrate a coincident microvillar actin bundle disruption and the perinuclear sequestration of F-actin in ATP-depleted proximal tubular cells.

ATP depletion in rat cholangiocytes leads to marked internalization of membrane proteins.

Intrahepatic bile ducts (BD) are a critical target of injury in the postischemic liver. Decreased vascular perfusion causes characteristic changes in the morphology of the ductular epithelia including a loss of secondary membrane structures and a decrease in plasma membrane surface area. Using adenosine triphosphate (ATP) depletion of cultured normal rat cholangiocytes (NRC) to model ischemic ducts, the present studies examined the fate of apical membrane proteins to determine whether membrane recycling might contribute to rapid functional recovery. Apical proteins, including gamma-glutamyl transpeptidase (GGT), Na(+)-glucose cotransporter (SGLT1), and apically biotinylated proteins, were not shed into the luminal space during ATP depletion. Instead, labeling of surface proteins after ATP depletion showed a significant decrease in GGT and SGLT1, consistent with membrane internalization. Similarly, z-axis confocal microscopy of biotinylated apical proteins also showed protein internalization. During ATP recovery, SGLT1 transport activity remained profoundly depressed even after 24 hours of recovery, indicating that the function of the internalized apical proteins is not rapidly recovered. These studies suggest that the membrane internalization in ATP-depleted cholangiocytes is a unidirectional process that contributes to prolonged functional deficits after restoration of normal cellular ATP levels. This sustained decrease in transport capacity may contribute to the development of ductular injury in postischemic livers.

The lipid products of phosphoinositide 3-kinase contribute to regulation of cholangiocyte ATP and chloride transport.

ATP stimulates Cl(-) secretion and bile formation by activation of purinergic receptors in the apical membrane of cholangiocytes. The purpose of these studies was to determine the cellular origin of biliary ATP and to assess the regulatory pathways involved in its release. In Mz-Cha-1 human cholangiocarcinoma cells, increases in cell volume were followed by increases in phophoinositide (PI) 3-kinase activity, ATP release, and membrane Cl(-) permeability. PI 3-kinase signaling appears to play a regulatory role because ATP release was inhibited by wortmannin or LY294002 and because volume-sensitive current activation was inhibited by intracellular dialysis with antibodies to the 110 kDa-subunit of PI 3-kinase. Similarly, in intact normal rat cholangiocyte monolayers, increases in cell volume stimulated luminal Cl(-) secretion through a wortmannin-sensitive pathway. To assess the role of PI 3-kinase more directly, cells were dialyzed with the synthetic lipid products of PI 3-kinase. Intracellular delivery of phosphatidylinositol 3, 4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate activated Cl(-) currents analogous to those observed following cell swelling. Taken together, these findings indicate that volume-sensitive activation of PI 3-kinase and the generation of lipid messengers modulate cholangiocyte ATP release, Cl(-) secretion, and, hence, bile formation.

Reorganization of cholangiocyte membrane domains represents an early event in rat liver ischemia.

Cholangiocytes contribute significantly to bile formation through the vectorial secretion of water and electrolytes and are a focal site of injury in a number of diseases including liver ischemia and post-transplantation liver failure. Using ischemia in intact liver and adenosine triphosphate (ATP) depletion in cultured cells to model cholangiocyte injury, these studies examined the effects of metabolic inhibition on cholangiocyte viability and structure. During 120 minutes of ischemia or ATP depletion, cell viability and tight junctional integrity in cholangiocytes were maintained. However, both the in vivo and in vitro models displayed striking alterations in the secondary structure of the plasma membrane. After 120 minutes, the basolateral (BL) interdigitations were diminished and the apical (Ap) microvilli were significantly decreased in number. The BL and Ap membrane surface areas decreased by 42 +/- 8% and 63 +/- 2%, respectively. Despite these changes, F-actin remained predominantly localized to the membrane domains. In contrast, in a time course that paralleled the loss of microvilli, the actin-membrane linking protein ezrin progressively dissociated from the cytoskeleton. These studies indicate that cholangiocyte ATP depletion induces characteristic, domain-specific changes in the plasma membrane and implicate alterations in the membrane-cytoskeletal interactions in the initiation of the changes. Pending the re-establishment of the differentiated domains, the loss of specific secondary structures may contribute to impaired vectorial bile duct secretion and postischemic cholestasis.

Distribution of epithelial ankyrin (Ank3) spliceoforms in renal proximal and distal tubules.

In diverse cell types, ankyrin tethers a variety of ion transport and cell adhesion molecules to the spectrin-based membrane skeleton. In the whole kidney, epithelial ankyrin (Ank3) is the predominantly expressed ankyrin and is expressed as distinct spliceoforms. Antibodies against a portion of the Ank3 regulatory domain detected four major spliceoforms at 215, 200, 170, and 120 kDa. Immunoblotting of the renal cortex, which is 80% proximal tubule (PT), detected all four spliceoforms but showed significantly diminished Ank3(200/215). To determine the Ank3 spliceoforms present in the mouse PT cells, PT fragments were purified to 100% from the renal cortex. Isolation was performed by incubating cortical tubule segments with fluorescein and isolating the fluorescein-laden PT fragments or fluorescein-deplete non-PT (distal) fragments under fluorescence microscopy. Distal tubule (DT) fragments displayed abundance of the Ank3(200/215) but no Ank3(170) or Ank3(120). Isolated PT segments contained all four spliceoforms but dramatically diminished Ank3(200/215). These larger spliceoforms bind Na-K-ATPase in diverse cell types. Densitometric analysis of Ank3(200/215) and Na-K-ATPase abundance measured a lower Ank3(200/215)-to-Na-K-ATPase ratio in the PT vs. the renal cortex. These proximal vs. distal differences in Ank3 spliceoforms were displayed in LLC-PK1 cells, a proximal cell line, and MDCK cells, a distal cell line. The lower PT content of Ank3(200/215) suggests Na-K-ATPase in PT may be organized differently than in DT. Likely reflecting their cell-specific organization, regulation, and function, these studies indicate the different renal cell types express distinct Ank3 spliceoforms.

Loss of cytoskeletal support is not sufficient for anoxic plasma membrane disruption in renal cells.

The goal of this study was to determine whether anoxic membrane disruption is initiated by loss of cytoskeletal support in rabbit renal proximal tubules (PT). We specifically tested 1) whether cytoskeletal perturbation affects membrane integrity under normoxia, 2) whether cytoskeletal perturbation potentiates anoxic membrane damage, and 3) whether the membrane protection by glycine depends on cytoskeletal integrity. Cytoskeletal perturbation was achieved with 10 microM cytochalasin D (CD) because it selectively disturbs F-actin organization and has similar effects as anoxia on the cytoskeleton of PT. During normoxia, CD caused decreased basal F-actin content, microvillar breakdown, and membrane-cytoskeleton dissociation, as revealed by the use of laser tweezers. However, membrane integrity was not altered by CD, as monitored by lactate dehydrogenase release. CD pretreatment of PT did not potentiate anoxic membrane damage. Finally, plasma membrane protection by glycine during anoxia remained in CD-pretreated PT despite loss of cytoskeletal support. These results demonstrate that loss of cytoskeletal support is not sufficient for anoxic plasma membrane disruption.

Loss of plasma membrane structural support in ATP-depleted renal epithelia.

Renal ischemia induces cytoskeletal alterations, membrane perturbations, including bleb formation, and ultimately membrane lysis. The mechanisms that underlie these alterations are largely unknown. Through the use of isolated rat renal proximal tubule fragments and calibrated micropipette techniques, two potential mechanisms for membrane bleb formation during ATP depletion were examined: 1) decreased cytoskeletal retention of the plasma membrane and 2) increased intracellular pressure. Under control conditions, the pressure required to pull the membrane from the underlying cellular matrix was 73 +/- 10 kdyn/cm2. After 30 min of ATP depletion, this pressure was diminished by >95% and blebs began to emerge from the basal membrane. The intracellular pressure within these blebbed cells was only 0.08 +/- 0.02 kdyn/cm2. These observations indicate that, during ATP depletion, the strength of membrane retention diminished until the relatively low intracellular pressure was capable of driving membrane bleb formation. Cytochalasin D, which disrupts the actin cytoskeleton, decreased the strength of membrane retention by 65 +/- 7%. This suggests that, during ATP depletion, alterations of the actin cytoskeleton may mediate the loss of membrane retention.

Actin and villin compartmentation during ATP depletion and recovery in renal cultured cells.

ATP-depletion in renal cultured cells has been used as a model for studying various cytoskeletal and functional alterations induced by renal ischemia. This communication explores the reversibility of these effects utilizing a novel method [1] that depleted ATP (ATP-D) to 2% of control within 30 minutes and caused complete recovery (REC) of ATP in one hour. Under confocal microscopy, ATP-D (30 min) caused thinning of F-actin from the microvilli, cortical region, and basal stress fibers, with the concurrent appearance of intracellular F-actin patches. These changes were more pronounced after 60 minutes of ATP-D. One hour of REC following 30 minutes of ATP-D produced complete recovery of F-actin in each region of the cell. However, after 60 minutes of ATP-D, a heterogeneous F-actin recovery pattern was observed: almost complete recovery of the apical ring and microvilli, thinned cortical actin with occasional breaks along the basolateral membrane, and a dramatic reduction in basal stress fiber density. The time course of cortical actin and actin ring disruption and recovery coincided with a drop recovery in the transepithelial resistance and the cytoskeletal dissociation and reassociation of the Na,K-ATPase. Additionally, the microvilli retracted into the cells during ATP-D, a process that was reversed during REC. Triton extraction and confocal microscopy demonstrated that villin remained closely associated with microvillar actin during both ATP-D and REC. These distinctive regional differences in the responses of F-actin to ATP depletion and repletion in cultured renal epithelial cells may help to clarify some of the differential tubular responses to ischemia and reperfusion in the kidney.

ATP depletion: a novel method to study junctional properties in epithelial tissues. II. Internalization of Na+,K(+)-ATPase and E-cadherin.

MDCK and JTC cells were subjected to ATP depletion by treating the cells with 10 microM antimycin A and 10 mM 2-deoxyglucose. As visualized by confocal fluorescence microscopy, E-cadherin and Na+,K(+)-ATPase were rapidly internalized following depletion of the intracellular ATP stores. The time course of internalization was similar to the depolymerization of the cortical actin network and dissolution of the actin ring (see companion paper, this volume, pp. 3301-3313). Cell surface biotinylation was used to assay the amount of surface-accessible E-cadherin and Na+,K(+)-ATPase during ATP depletion. At 30 minutes of ATP depletion, 74% and 69% of E-cadherin and Na+,K(+)-ATPase were internalized, respectively, in MDCK cells. By 60 minutes of ATP depletion, internalization increased to 95% and 89%, respectively. The redistribution of both plasma membrane proteins was not microtubule dependent. Similar results were observed in JTC cells. Total biotinylated protein decreased by 67% and 82%, after 30 minutes and 60 minutes of ATP depletion, respectively. The E-cadherin internalization strongly suggests that disruption of adherens junctions occurred following ATP depletion. These results, along with the previously described loss of tight junction integrity, suggest that ATP depletion may be a useful method to study the assembly and disassembly of junctional complexes in epithelial cells.

Cytoskeletal dissociation of ezrin during renal anoxia: role in microvillar injury.

The association/dissociation of ezrin, a microvillar membrane-cytoskeleton linker, was studied to search for the initial step leading to anoxia-induced brush-border breakdown in a rabbit proximal tubule suspension. Electron microscopy studies display time-dependent damage to the microvilli during anoxia; immunoblots demonstrate the dissociation of ezrin from the cytoskeleton, reflected by the significant decrease in Triton X-100-insoluble ezrin from control (91%) to 39% after 30 min. Simultaneously, Triton X-100-soluble and extracellular ezrin increased with no change in total ezrin, Triton X-100 solubility of actin, or total intracellular protein. Parallel immunocytochemistry studies show diffusion of ezrin from the brush border, where ezrin is highly colocalized with F-actin during normoxia into the cytoplasm. Thirty minutes of reoxygenation following 30 min of anoxia causes recovery of the microvillar structure and reassociation of ezrin to the cytoskeleton and the brush border. Application of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (4 mM) or inhibition of intracellular calpain or calcineurin do not prevent the dissociation of ezrin during anoxia. We conclude that ezrin-cytoskeletal dissociation may initiate microvillar breakdown during anoxia via calcium-independent mechanisms.

Method for recovering ATP content and mitochondrial function after chemical anoxia in renal cell cultures.

Cultured renal cells provide a highly reproducible and malleable model to study cellular responses to metabolic perturbations. Nevertheless, there is currently no good method to achieve metabolic inhibition and complete recovery in cultured cells. This study describes a specific method for reversibly inhibiting both glycolytic and oxidative metabolism. Glycolysis was inhibited by removing all glycolytic substrates, and mitochondrial respiration was inhibited with rotenone, a site I inhibitor of the electron transport chain. Within 30 min, ATP values were decreased by 98%. Glycolysis was restored through the reintroduction of glucose. Oxidative metabolism was restored by the addition of heptanoate, a short odd-chain fatty acid, which supplies reducing equivalents to site II of the electron transport chain. Employing Madin-Darby canine kidney and LLC-PK1 cell lines, this protocol caused the immediate and complete recovery of mitochondrial respiration and, by 60 min, the complete recovery of cellular ATP levels. Application of this protocol should allow the investigation of the cellular effects and alterations that occur within cells recovering from sublethal energy depletion.

Degradation of spectrin and ankyrin in the ischemic rat kidney.

This study investigates ischemia-induced degradation of the spectrin-based cytoskeleton in rat brain, heart, and kidney. Spectrin, in conjunction with ankyrin, structurally supports the plasma membrane and sequesters integral membrane proteins. After 60 and 120 min of ischemia, brain tissue displayed both spectrin and ankyrin breakdown. The spectrin fragmentation pattern is similar to previously reported ischemia-induced calpain I proteolysis of spectrin in N-methyl-D-aspartate receptor-containing neurons. Ischemic heart tissue displayed no spectrin or ankyrin degradation. Ischemic renal tissue showed minimal breakdown of spectrin but a major loss of ankyrin (25%/30 min of ischemia) that was essentially complete after 120 min of ischemia. Interestingly, this profound loss of ankyrin in the intact ischemic kidney was not mimicked in three renal cell lines (MDCK, LLC-PK1, and JTC cell lines) exposed to chemical anoxia. Immunocytochemistry showed ankyrin was concentrated in thick ascending limb (cTAL) cells and, although delayed by 30 min, was lost at the same rate as measured by immunoblot analysis. Spectrin and Na(+)-K(+)-ATPase, which complex with ankyrin, were essentially unaffected by ischemia. Ankyrin degradation in cTAL cells correlated with the loss of basal infolding organization. In conclusion, the spectrin-based cytoskeleton is differentially targeted by ischemia-induced degradative processes in different in vivo tissues.

Minimal role of xanthine oxidase and oxygen free radicals in rat renal tubular reoxygenation injury.

The role of xanthine oxidase and oxygen free radicals in postischemic reperfusion injury in the rat kidney remains controversial. Proximal tubules, the focal segment affected by ischemic renal injury, were isolated in bulk, assayed for xanthine oxidase activity, and subjected to 60 min of anoxia or hypoxia and 60 min of reoxygenation to evaluate the participation of xanthine oxidase and oxygen radicals in proximal tubule reoxygenation injury. The total xanthine oxidase in isolated rat proximal tubules was 1.1 mU/mg of protein, approximately 30% to 40% of the activity found in rat intestine and liver. Lactate dehydrogenase release, an indicator of irreversible cell damage, increased substantially during anoxia (39.8 +/- 2.3 versus 9.8 +/- 1.8% in controls) with an additional 8 to 12% release during reoxygenation. Addition of 0.2 mM allopurinol, a potent xanthine oxidase inhibitor, and dimethylthiourea, a hydroxyl radical scavenger, failed to protect against the reoxygenation lactate dehydrogenase release. Analysis of xanthine oxidase substrate levels after anoxia and flux rates during reoxygenation indicates that hypoxanthine and xanthine concentrations are in a 15-fold excess over the enzyme Km and 0.3 mU/mg of protein of xanthine oxidase activity exists during reoxygenation. Hypoxic tubule suspensions had a minimal lactate dehydrogenase release during hypoxia and failed to demonstrate accelerated injury upon reoxygenation. In conclusion, although xanthine oxidase is present and active during reoxygenation in isolated rat proximal tubules, oxygen radicals did not mediate reoxygenation injury.

Pharmacology of Acorus calamus L.

Water soluble dried powder of alcoholic extract of roots and rhizomes of A. calamus L. was used. The in vivo experiments involved strychnine convulsant activity in frogs, spontaneous motor activity and amphetamine hyperactivity in mice, pentobarbitone sleeping-time in rats and local anaesthetic activity in guinea pigs and rabbits. Frog skeletal muscle and heart preparations and rat phrenic nerve diaphragm constituted the in vitro experiments. Plant extracts at 10, 20 mg/kg ip did not afford protection to strychnine (1,5,2.5 mg/kg) induced convulsions and same effect was found on acetylcholine induced contractions of rectus muscle except that it inhibited caffeine citrate contractions in frog. At 1, 10 and 100 micrograms/ml doses, it caused negative iono- and chronotropic effects in frogs. Dosages of 10, 25, 50 mg/kg ip of herbal extract antagonize spontaneous motor activity and also amphetamine induced hyperactivity in mice. It was less potent than chloropromazine, though exerts sedative and tranquilizing action. Local anaesthetic activity was found to be absent at 0.5 and 1% dose levels.

Possible role of dopamine in the growth inhibitory effect of pentazocine.

Growth inhibitory effect of pentazocine was studied in young rats for a duration of 4 weeks. The animals were subjected to various drug treatments, namely (1) distilled water 0.5 ml/kg s.c.; (2) pentazocine 10 mg/kg s.c.; (3) pentazocine 10 mg/kg s.c. plus metoclopramide 10 mg/kg s.c., and (4) pentazocine 10 mg/kg s.c. plus l-dopa 100 mg/kg s.c. Body weight was taken as a measure for the assessment of growth rate. The results of group 2 were compared with group 1 and that of groups 3 and 4 were compared with group 2. Pentazocine alone markedly inhibited the growth from the second to fourth weeks of treatment. Dopamine antagonist metoclopramide has almost completely prevented the growth inhibitory effect of pentazocine. Combination of l-dopa with pentazocine produced a significant growth inhibition in the first week but subsequently this effect was significantly less marked as compared to that of pentazocine alone. The growth inhibitory effect of pentazocine may possibly be mediated through alterations in dopaminergic mechanisms which are known to exert controlling influence on the release of several of the pituitary hormones.