Vasopressin-stimulated Ca2+ spiking in vascular smooth muscle cells involves phospholipase D

Physiological concentrations of [Arg(8)]vasopressin (AVP; 10-500 pM) stimulate oscillations of cytosolic free Ca2+ concentration (Ca2+ spikes) in A7r5 vascular smooth muscle cells. We previously reported that this effect of AVP was blocked by a putative phospholipase A2 (PLA2) inhibitor, ONO-RS-082 (5 microM). In the present study, the products of PLA2, arachidonic acid (AA), and lysophospholipids were found to be ineffective in stimulating Ca2+ spiking, and inhibitors of AA metabolism did not prevent AVP-stimulated Ca2+ spiking. Thin layer chromatography was used to monitor the release of AA and phosphatidic acid (PA), which are the products of PLA2 and phospholipase D (PLD), respectively. AVP (100 pM) stimulated both AA and PA formation, but only PA formation was inhibited by ONO-RS-082 (5 microM). Exogenous PLD (type VII; 2.5 U/ml) stimulated Ca2+ spiking equivalent to the effect of 100 pM AVP. AVP stimulated transphosphatidylation of 1-butanol (a PLD-catalyzed reaction) but not 2-butanol, and 1-butanol (but not 2-butanol) completely prevented AVP-stimulated Ca2+ spiking. Protein kinase C (PKC) inhibition, which completely prevents AVP-stimulated Ca2+ spiking, did not inhibit AVP-stimulated phosphatidylbutanol formation. These results suggest that AVP-stimulated Ca2+ spiking depends on activation of PLD rather than PLA2 and that PKC activation may be downstream of PLD in the signaling cascade.     

Atrial natriuretic peptide inhibits the phosphoinositide hydrolysis in murine Leydig tumor cells

The ability of ANP to inhibit the hydrolysis of phosphoinositides was examined in [³H] myoinositol-labeled intact murine Leydig tumor (MA-10) cells. Arginine vasopressin (AVP) stimulated the formation of inositol monophosphate (IP₁), inositol bisphosphate (IP₂), and inositol trisphosphate (IP₃) both in a time- and dose- dependent manner in MA-10 cells. ANP inhibited the AVP-induced formation of IP₁, IP₂, and IP₃ in these cells. The inhibitory effect of ANP on the AVP-stimulated formation of IP₁, IP₂, and IP₃ accounted for 30%, 38% and 42%, respectively, which was observed at the varying concentrations of AVP. ANP caused a dose-dependent attenuation in AVP-stimulated production of IP₁, IP₂ and IP₃ with maximum inhibition at 100 nM concentration of ANP. The production of inositol phosphates was inhibited in the presence of 8- bromo cGMP in a dose-dependent manner, whereas dibutyryl-cAMP had no effect on the generation of these metabolites. The LY 83583, an inhibitor of guanylyl cyclase and cGMP production, abolished the inhibitory effect of ANP on the AVP-stimulated production of inositol phosphates. Furthermore, 10 M LY 83583 also inhibited the ANP-stimulated guanylyl cyclase activity and the intracellular accumulation of cGMP by more than 65–70%. The inhibition of eGMP-dependent protein kinase by H-8, significantly restored the levels of AVP-stimulated inositol phosphates in the presence of either ANP or exogenous 8-bromo cGMP. The results of this study suggest that ANP exerts an inhibitory effect on the production of inositol phosphates in murine Leydig tumor (MA-10) cells by mechanisms involving cGMP and cGMP-dependent protein kinase.Established Investigator of the American Heart Association     

Activation of phospholipase C and prostaglandin synthesis by [arginine]vasopressin in cultures.

[Arginine]vasopressin (AVP) stimulates maximal prostaglandin E2 production in cultured rat renal mesangial cells within 2 min. As early as 10s after addition of AVP (10(-6)M) a significant loss of radioactivity from phosphatidylinositol 4,5-bisphosphate but not from phosphatidylinositol 4-phosphate and phosphatidylinositol was observed in cells prelabelled with 32Pi. Cells labelled with [14C]arachidonic acid showed an increase of label in 1,2-diacylglycerol after 15 s and in phosphatidic acid after 30 s upon stimulation with AVP. Pretreatment of the cells with indomethacin (10(-5)M) did not abolish the effect of AVP on the increased labelling of phosphatidic acid.     

Ca2+ influx stimulated by vasopressin is mediated by phosphoinositide hydrolysis in rat smooth muscle cells

The mechanism of Ca2+ influx stimulated by arginine vasopressin (AVP) was studied in cultured rat smooth muscle cells. AVP stimulated 45Ca2+ influx even in the presence of nifedipine, a Ca2+ antagonist that inhibits voltage-dependent Ca2+ channel. NaF, a GTP-binding protein activator, mimicked the AVP-stimulated 45Ca2+ influx. The 45Ca2+ influx stimulated by a combination of AVP and NaF was not additive. The affinity of AVP receptor was decreased by guanosine 5'-O-(3-thiotriphosphate). Pertussis toxin failed to affect the AVP-stimulated 45Ca2+ influx. AVP did not stimulate cAMP production, but increased inositol trisphosphate generation. Both AVP-stimulated 45Ca2+ influx and inositol trisphosphate generation were inhibited by neomycin, a phospholipase C inhibitor, in a dose-dependent manner, and the patterns of both inhibitions were similar. These results suggest that, in rat smooth muscle cells, AVP-stimulated Ca2+ influx is mediated exclusively through phosphoinositide hydrolysis.     

Glucose-stimulated protein phosphorylation in the pancreatic islet

The effect of inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism on steroidogenesis in rat testis Leydig cells and rat tumour Leydig cells has been investigated. In the presence of nordihydroguaiaretic acid [NDGA; 4,4'-(2,3- dimethylbutan -1,4- diyl )bis[1,2- benzendiol ]], 5,8,11,14-eicosatetraynoic acid (ETYA), BW 755C [3-amino-1-[3-(trifluoromethyl)phenyl]-2-pyrazoline hydrochloride] and benoxaprofen [ Opren ; 2-(2-p-chlorophenyl- benzoxazol -5-yl)propionic acid)] (which inhibit lipoxygenase activity), but not indomethacin and aspirin (which inhibit cyclo-oxygenase activity), a dose-related inhibition of lutropin (LH)-stimulated testosterone and pregnenolone production was obtained (ID50 values of 2.5, 30, 25 and 30 microM for NDGA, ETYA, BW 755C and benoxaprofen were obtained, respectively). BW 755C and benoxaprofen had no significant effect on LH-stimulated cyclic AMP production except at the highest concentrations examined (330 and 380 microM, respectively), whereas NDGA and ETYA inhibited LH-stimulated cyclic AMP production in a dose-dependent manner (ID50 7.0 and 22 microM respectively). However, NDGA and ETYA also caused a dose-dependent inhibition of dibutyryl cyclic AMP-stimulated testosterone and pregnenolone production. The metabolism of exogenous ( 22R )-hydroxycholesterol or pregnenolone to testosterone by Leydig cells was not inhibited by either NDGA, ETYA or indomethacin. At low concentrations of NDGA and ETYA a significant increase in the conversion of both pregnenolone and ( 22R )-hydroxycholesterol to testosterone was obtained. Studies in which the metabolism of [14C]arachidonic acid by purified rat tumour Leydig cells was investigated indicate that products are formed by tumour Leydig cells that have similar mobilities in a thin layer chromatography system to 5-L-hydroxy-6,8,11,14-eicosatetraenoic acid, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and leukotriene B4. The formation of these products was inhibited to varying degrees by NDGA, BW 755C and benoxaprofen but not by aspirin and indomethacin. These studies demonstrate for the first time that inhibition of lipoxygenase activity but not cyclo-oxygenase activity causes an inhibition of LH- and dibutyryl cyclic AMP-stimulated steroid production and suggest a stimulatory role for products of the lipoxygenase pathway of arachidonic acid metabolism in steroidogenesis. The site of this stimulation is apparently distal to the production of cyclic AMP and before the side chain cleavage of cholesterol.     

Evidence that the effects of arginine-8-vasopressin (AVP) on pituitary corticotropin (ACTH) release are mediated by a novel type of receptor

Ovine corticotropin releasing factor (oCRF-41) and AVP act synergistically to stimulate pituitary ACTH secretion. In the present study we have investigated whether the effect of AVP, either in the presence or in the absence of oCRF-41 (0.5 nmol/l), could be blocked by V1 (pressor)-antagonists. Furthermore, oxytocin, and [1-deamino,8-D-arginine] vasopressin (dDAVP) were tested for their ability to release ACTH. All experiments were carried out in vitro, using segments of rat anterior pituitary glands. The V1-antagonist [1-deamino,penicillamine(o-methyl-tyrosine)]AVP inhibited ACTH release induced by AVP or AVP + oCRF-41. However, it also had some agonistic activity which was more pronounced in the presence of oCRF-41. An equally potent V1-antagonist, [1-beta-mercapto-beta, beta-cyclopentamethyleneproprionic acid (o-methyl-tyrosine)]AVP, failed to inhibit AVP-stimulated ACTH secretion, and also had weak agonist potency. The relatively selective V2 (antidiuretic)-agonist dDAVP was 20-30 fold less potent than AVP. Oxytocin, a weak V1- and V2-agonist was only 4-8 fold less potent than AVP. These data are compatible with the suggestion that AVP receptors on pituitary corticotrope cells are neither classical V1- nor V2-receptors.     

Bradykinin effects on phospholipid metabolism and its relation to arachidonic acid turnovers in neuroblastoma × glioma hybrid cells (NG 108-15)

In neuroblastoma × glioma hybrid cells (NG 108-15) labelled with [³²P]-trisodium phosphate, [³H]-inositol and [¹⁴C]-arachidonic acid, bradykinin stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) while it had no effect on the release of [¹⁴C]-arachidonic acid (AA). The effect on PIP, was time- and dose-dependent with a maximal effect on [³H]-inositol- and [³²P]-labelled cells after 10–30 s of stimulation with 10⁻⁶ M bradykinin. However, the hydrolysis of [¹⁴C]-AA labelled PIP₂ was delayed compared to the effect on [³H]- and [¹⁴C]-PIP₂ and was not detectable until after 60 s of stimulation. Bradykinin stimulation resulted in an increased formation of [³H]-inositol phosphates (IP) and [³²P]- and [¹⁴P]- and [¹⁴C]-phosphatidic acid (PA) but the time course for PA formation did not allow the time-course for PIP₂ hydrolysis. A reduced labelling of [²³P]- and [¹⁴C]-phosphatidylcholine was also found in stimulated cells suggesting that PA may derive from other sources than PIP₂. In conclusion, our results indicate that bradykinin activates phospholipase C, but not phospholipase A₂, in NG 108-15 cells.     

Effect of a new V1 antagonist (OPC-21268) on vascular action of vasopressin in cultured rat vascular smooth muscle cells.

The effect of 1-(1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl-3,4-dihydro-2(1 H)- quinolinone) (OPC-21268) on vascular action of arginine vasopressin (AVP) was examined in cultured rat vascular smooth muscle cells (VSMC) by the measurement of cytosolic free calcium concentration [( Ca2+]i) and the AVP V1 receptor study. The preincubation of cells with OPC-21268 for 10 min inhibited the AVP-induced mobilization of [Ca2+]i in a dose-dependent manner but did not affect the angiotensin II-induced mobilization of [Ca2+]i. The receptor study revealed that OPC-21268 blocks the binding of AVP to the receptor in VSMC in a similar way to the V1 structural antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid)-2-O-methyltyrosine]AVP: d(CH2)5Tyr(Me)AVP. Lineweaver-Burk plot showed that OPC-21268 is the competitive AVP V1 receptor antagonist. These results therefore indicate that OPC-21268 specifically blocks the vascular action of AVP mediated through the competitive inhibition of AVP binding to the receptors in VSMC.     

Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.

Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of phospholipase D (PLD) activation, was monitored. ET-1 stimulated much greater PEt formation in the PKC overexpressing cells. ET-1 action was dose-dependent with a half-maximal effect at 1.0 x 10(-9) M. With increasing ethanol concentrations, [3H]PEt formation increased at the expense of [3H]phosphatidic acid (PA). Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA accumulation and decreased [3H]diacylglycerol (DAG) formation. These data are consistent with the formation of [3H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10(-9) M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.     

Vasopressin-induced increases in cellular free calcium concentration measured in single cells of rat renal papillary collecting tubule

We determined the cellular free calcium concentration [Ca2+]i in response to arginine vasopressin (AVP) using single cells of cultured rat renal papillary collecting tubule cells. AVP at a concentration of 1 x 10(-10) M or higher significantly increased [Ca2+]i in a dose-dependent manner. The prompt increase in [Ca2+]i induced by AVP was completely blocked by the V1V2 antagonist, but not by the V1 antagonist. Also, an antidiuretic agonist of 1-deamino-8-D-arginine vasopressin (dDAVP) increased [Ca2+]i, which was blocked by the pretreatment with the V1 V2 antagonist. An AVP-induced increase in [Ca2+]i was still demonstrable in cells pretreated with Ca2(+)-free medium containing 1 x 10(-3) M EGTA, or a blocker of cellular Ca2+ uptake, 5 x 10(-5) M verapamil. These results indicate that AVP increases [Ca2+]i through the V2 receptor in renal papillary collecting tubule cells where cAMP is a well-known second messenger for AVP, and that cellular free Ca2+ mobilization depends on both the intracellular and extracellular Ca2+.     

AVP receptors of mouse Leydig cells are regulated by LH and E2 and influenced by experimental cryptorchidism

Exposure of pubertal mouse Leydig cells for 24 h to increasing concentrations (1-100 ng/ml) of LH elicited a dose-dependent decrease in AVP receptor content. Maximal reduction (50%) was obtained at a dose of 10 ng/ml LH. A similar treatment applied to adult Leydig cells did not influence AVP receptor density. Treatment of adult Leydig cells for 24 h by E2 (5-500 ng/ml) resulted in a dose-dependent increase in AVP receptor content. About 50% increase was achieved with 500 ng/ml E2. AVP receptor content in pubertal Leydig cells was not modified irrespective of the concentration of E2 tested. These changes in AVP receptor number were well correlated with the response of Leydig cells to AVP (10(-6) M) in terms of testosterone production. 2 weeks bilateral cryptorchidism resulted in reduction of testicular weight, circulating testosterone levels associated with a marked rise in Leydig cell AVP receptor density with no change of affinity. Testosterone production by Leydig cells from cryptorchid testes in response to AVP (10(-6) M) or hCG (100 ng/ml) stimulation was reduced compared to that of control Leydig cells. This study provides new arguments supporting the concept that AVP could be involved in local regulation of testicular steroidogenesis.     

Effect of vasopressin on Na+ kinetics in cultured rat vascular smooth muscle cells

The effect of arginine vasopressin (AVP) on Na+ kinetics was examined in cultured rat vascular smooth muscle cells (VSMC) and rat renal papillary collecting tubule cells (RPCT) by the direct measurement of intracellular sodium concentration [(Na+]i) using fluorescence dye; SBFI. AVP increased [Na+]i in a dose-dependent manner at a concentration of 10(-9) M or higher in rat VSMC but did not affect [Na+]i in rat RPCT. The calcium (Ca2+)-free solution completely blocked the increasing effect of AVP on [Na+]i in rat VSMC. A Ca2+ ionophore, ionomycin (1-2 x 10(-6) M) increased [Na+]i both in rat VSMC and RPCT. The Ca2(+)-free solution abolished the ionomycin-increased [Na+]i both in rat VSMC and RPCT. These results therefore indicate that after binding the V1 receptor AVP increases [Na+]i mediated through an increase in cellular Ca2+ uptake in VSMC.     

Time-related effects of arginine vasopressin on steroidogenesis in cultured mouse Leydig cells

The dose and time treatment effects of arginine vasopressin (AVP) on basal and hCG-stimulated testosterone accumulation by purified mouse Leydig cells in primary culture were examined. Pretreatment for 24 h of Leydig cells with AVP caused a stimulation of the acute (3 h) basal testosterone accumulation. In these conditions, progesterone accumulation was also increased. The stimulatory effect of AVP (10(-11)-10(-5) M) on testosterone accumulation was dose-dependent and as little as 10(-11) M-AVP caused significant stimulation whilst maximal effect was achieved with 10(-7) M. Oxytocin (10(-6) M) also showed a stimulation of testosterone accumulation in basal conditions, but the other peptides tested at the same concentration (neurotensin, somatostatin and substance P) did not have any effect. When Leydig cells were exposed to AVP for a longer period (48 or 72 h), the increase in basal testosterone accumulation disappeared. AVP treatment of Leydig cells for 72 h led to a significant and dose-dependent reduction in the hCG-responsiveness without altering the slope of the hCG dose-response curve. This inhibitory effect, which was also observed when AVP-pretreated Leydig cells were acutely challenged for 3 h with 8-bromo-cAMP, was accompanied by a concomitant increase in progesterone accumulation. These results indicate that AVP can exert a dual effect on mouse Leydig cells: stimulatory on basal testosterone accumulation during short-term exposure (24 h) and inhibitory on the response to hCG stimulation after long-term treatment (72 h). They provide additional evidence that neurohypophysial peptides directly affect Leydig cell steroidogenesis.     

Vasopressin stimulates tyrosine phosphorylation by activation of PKC in the rat smooth muscle cell line, A-10

Arginine vasopressin (AVP)-induced tyrosine phosphorylation was studied in a rat smooth muscle cell line, A-10, by western blotting, using a monoclonal antibody against phosphotyrosine. AVP stimulated the phosphorylation of several cellular proteins of molecular mass 60-130 kDa in a time- and dose-dependent manner. Phosphorylation was mediated largely by V(1)receptor subtype since it was inhibited by selective V(1)antagonist and was only partially elicited by the V(2)agonist, desmopressin. Heterotrimeric G-proteins seemed to be involved in the phosphorylation mechanism because fluoraluminates, an activator of heterotrimeric G-proteins (and thus an uncoupler of the receptor-G-protein interaction) inhibited the AVP-induced phosphorylation. The protein kinase C (PKC) inhibitors: staurosporine, H7 and GF109203X are able to block the AVP-stimulated phosphorylation. The last of these has been shown to be one of the most selective inhibitors of PKC. These results indicate that PKC is upstream of the phosphorylation, a motion which is supported by the fact that the AVP-stimulated phosphorylation was downregulated by phorbol esters.     

Inhibition of prostaglandin E2 synthesis by a blocker of epithelial chloride channels

Arginine-vasopressin (AVP) elicits a variety of responses in cultured rat mesangial cells, among them stimulation of prostaglandin biosynthesis and activation of Cl- channels. AVP produced an 11-fold increase over basal levels in prostaglandin E2 release from cultured mesangial cells. This response was completely inhibited by 25 microM indomethacin and 82 +/- 5% inhibited by 25 microM 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) which is a potent blocker of epithelial Cl- channels. The IC50 for NPPB inhibition of prostaglandin E2 release was 8 microM. Indomethacin and NPPB at 25 microM also inhibited AVP-stimulated cellular accumulation of prostaglandin E2 by 98% and 79 +/- 7% respectively. The inhibitory effect of NPPB was not due to interference with the cellular response to AVP since at 50 microM it did not block AVP-stimulated release of arachidonate metabolites from cells metabolically labeled with [3H]-arachidonic acid. It is suggested that NPPB inhibition of prostaglandin E2 synthesis is at the cyclooxygenase level on the basis of its structural similarity to the fenamic acid type of cyclooxygenase inhibitors.     

Effect of inhibition of Na+/K(+)-ATPase on the vasopressin-induced increase in intracellular Na+ concentration in vascular smooth muscle cells.

The effect of inhibition of Na+/K(+)-ATPase by ouabain on the arginine vasopressin (AVP)-induced increase in intracellular Na+ concentration [( Na+]i) was examined in cultured rat vascular smooth muscle cells (VSMC) by the direct measurement of [Na+]i using a fluorescent indicator dye. AVP at a concentration of 1 x 10(-9) M or higher increased [Na+]i in a dose-dependent manner in cultured rat VSMC. The preincubation of cells with 1 x 10(-4) M ouabain for 1 hr at 37 degrees C did not affect the basal [Na+]i but enhanced the 1 x 10(-6) M AVP-induced increase in [Na+]i. The preincubation was not necessary because similar results were obtained after the simultaneous administration of AVP and ouabain. The treatment with ouabain did not affect the intracellular pH changes induced by AVP. These results therefore indicate that the inhibition of Na+/K(+)-ATPase enhances the AVP-induced increase in [Na+]i by decreasing cellular Na+ efflux in cultured rat VSMC.     

Arginine vasopressin inhibits apoptosis of rat glomerular mesangial cells via V1a receptors

Arginine vasopressin (AVP) promotes proliferation of glomerular mesangial cells. We examined whether AVP modulates an apoptosis of cultured rat glomerular mesangial cells at 3-17th passages. The agarose gel electrophoresis demonstrated that AVP attenuated a ladder formation stimulated by the serum deprivation. The quantitation of oligonucleosomes by ELISA also showed that AVP suppressed the serum deprivation-induced apoptosis. Such an antiapoptotic effect of AVP was dose-dependent. An AVP V1a receptor antagonist, d(CH2)5Tyr(Me)AVP, abolished the antiapoptotic effect of AVP. The inhibitory effect of AVP on the apoptosis was reduced by staurosporine and mimicked by phorbol-12-myristate-13-acetate. These results suggest that AVP inhibits serum deprivation-induced apoptosis of glomerular mesangial cells via V1a receptor-protein kinase C pathway.     

1 alpha,25-dihydroxyvitamin D3 modulation in lipid metabolism in established bone marrow-derived stromal cells, MC3T3-G2/PA6.

MC3T3-G2/PA6 (PA6) cells established from newborn mouse calvaria are preadipocytic stromal cells, which differentiate into adipocytes in response to glucocorticoids. We examined the effects of 1 alpha,25-dihydroxyvitamin D3[1 alpha,25(OH)2D3] on adipogenesis in PA6 cells. When PA6 cells were cultured with 10(-8) M dexamethasone, adipocytes containing oil red O-positive droplets first appeared on day 7 (3 days after confluence was attained) and the maximal synthesis of neutral lipids occurred on day 12. Simultaneous addition of 1 alpha,25(OH)2D3 at 10(-9)M completely blocked this dexamethasone-induced neutral lipid synthesis throughout the 14-day culture period. Dose-response studies of vitamin D3 derivatives showed that 1 alpha,25(OH)2D3 was the most potent in inhibiting neutral lipid synthesis in PA6 cells, followed by 1 alpha-hydroxyvitamin D3, 25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3, in that order. Dexamethasone greatly enhanced incorporation of [14C]-acetic acid into triacylglycerol in PA6 cells. The incorporation was markedly inhibited by the addition of 10(-9) M 1 alpha,25(OH)2D3. Instead, 1 alpha,25(OH)2D3 greatly increased incorporation of [14C]-acetic acid into phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, irrespective of the presence or absence of dexamethasone. These results suggest that 1 alpha,25(OH)2D3 modulation of lipid metabolism in bone marrow stromal cells is receptor mediated.     

Facilitation of phospholipase A2 activity by mastoparans, a new class of mast cell degranulating peptides from wasp venom

The effect of mastoparan, Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-LeuNH2, and related peptides on the release of arachidonic acid from egg yolk lecithin liposomes, rat peritoneal mast cells, and cultured human fibroblasts was studied. In unsonicated liposomes, labeled with 1-stearoyl-2[1-14C]arachidonyl-sn-glycero-3-phosphocholine, 5 X 10(-5) M mastoparan caused a 12-, 15-, and 50-fold increase in the production of arachidonic acid catalyzed by phospholipase A2 from bee venom, eastern diamondback rattlesnake and porcine pancreas, respectively. The stimulant effect of mastoparan and related peptides was dose-dependent and further enhanced by sonication of liposomes. In contrast, melittin, while stimulating the production of arachidonic acid by phospholipase from bee venom, was inactive with the rattlesnake and pancreatic enzymes. Melittin was also only weakly active with liposomes containing stearic acid in place of arachidonic acid. Like melittin, mastoparans stimulated phospholipase activity in tissue homogenates and caused a dose-dependent release of arachidonic acid from rat peritoneal mast cells and cultured human fibroblasts prelabeled with [14C]arachidonic acid. The heptapeptide fragments mastoparan 1-7 and mastoparan 8-14, and succinylated mastoparan were ineffective. The results suggest that mastoparan and related peptides in insect venoms act, at least in part, by stimulating phospholipase activity.     

Effect of 3,5,3'-triiodo-L-thyronine on amino acid accumulation and membrane potential in Sertoli cells of the rat testis

The purpose of this study was to investigate the effect of T3 on amino acid accumulation and on the membrane potential of Sertoli cells of immature rat testes. Testes of pre-pubertal and pubertal rats were pre-incubated (30 min) in Krebs-Ringer bicarbonate buffer and incubated in the presence of [14C]methylaminoisobutyric acid with and without T3 for 15, 45 and 60 min. The hormone (10(-6) M and 10(-7) M) significantly stimulated amino acid accumulation in 6 and 13-day old rat testes but did not have any effect in neonatal and pubertal animals. T3 produced a dose-dependent hyperpolarizing effect at concentrations of 10(-6) M, 10(-5) M, 2 x 10(-5) M and 10(-4) M. We conclude that T3 induces a membrane hyperpolarization in Sertoli cells and stimulates amino acid accumulation in immature rat testes, demonstrating that the hormone has a rapid plasma membrane action.