Vascular responsiveness to phorbol esters in coarctation-hypertensive rats

Recent observations suggest that a phospholipid-sensitive, calcium-dependent protein kinase affects the contractile responses of vascular smooth muscle. Protein kinase C activators such as the tumor-promoting phorbol esters have been used as tools to study protein kinase C function in various intact cells. The present study characterizes vascular reactivity to protein kinase C activation in rats made hypertensive by coarctation of the abdominal aorta. Thoracic aortic strips from hypertensive rats developed greater force than arteries from normotensive rats in response to the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Thoracic aortae from hypertensive rats were more responsive (lower threshold dose) to the phorbol ester than those from normotensive rats. Additionally, arteries from hypertensive rats were more responsive to the contractile effects of mezerein, a non-phorbol ester activator of protein kinase C. Removal of the endothelium did not eliminate the difference in responsiveness to TPA in thoracic aortae from normotensive and hypertensive rats. The threshold dose of TPA in abdominal aortae from hypertensive rats was not different from that in normotensive rats. However, the maximal response to 10(-6) mol/l TPA after 60 min in abdominal aortae from hypertensive rats was significantly less than that in aortae from normotensive rats. Thus, contractile responses to TPA appear to be influenced by arterial pressure per se. The inhibitory effects of the calcium antagonist, verapamil, in thoracic aortae from hypertensive rats were greater than in those from normotensive rats. Verapamil inhibited TPA-induced contractions in abdominal aortae from hypertensive rats to the same extent as in those from normotensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of lead on rat thoracic aorta contraction and relaxation

Low levels of lead, but not high levels, produce hypertension. This mystery has not yet been resolved. In this study we compared the in vitro vasoresponsiveness in rat thoracic aorta to low dose (10⁻⁸ mol/L) and high dose (10⁻⁵ mol/L and 10⁻⁴ mol/L) lead acetate. In addition to the direct response to lead, we examined reactivity to norepinephrine, acetylcholine, isoproterenol, phorbol ester, and calcium in the presence and absence of lead. Neither low-dose nor high-dose lead directly affected aortic contractile or relaxant responses. However, lead, only at the highest concentration (10⁻⁴ mol/L), increased the contractions to calcium at all submaximal calcium concentrations. We conclude that low-dose lead must increase blood pressure indirectly through a humoral effect. The reasons for the failure of high-dose lead to influence blood pressure remain to be explored.     

Contrasting effects of phorbol esters on serotonin- and vasopressin-evoked contractions in rat aorta and small mesenteric artery.

Phorbol esters, which activate protein kinase C, modulate vasoconstrictor-induced tension in vascular smooth muscle. We examined the effects of phorbol esters (phorbol 12,13-dibutyrate [PDBu] and 12-O-tetradecanoylphorbol 13-acetate [TPA]) on receptor agonist (serotonin [5-HT] and arginine vasopressin [AVP])-, high K(+)-, and caffeine-induced contractions in rings of rat aorta and a small (second-order) branch of the superior mesenteric artery (SMA). PDBu and TPA significantly augmented agonist-evoked contractions in aorta but diminished those in SMA. For example, 30 nM PDBu increased 5-HT- and AVP-evoked contractions 2.0-2.5-fold in aorta (p less than 0.01) but decreased 5-HT- and AVP-induced contractions by 40-60% in SMA (p less than 0.01). In contrast, PDBu and TPA amplified high K(+)- and 10 mM caffeine-induced contractions in both aorta and SMA. Augmentation of agonist-induced contractions by PDBu was greater in endothelium-denuded aorta than in intact aorta. Two protein kinase C antagonists, H-7 and staurosporine, inhibited 5-HT-evoked contractions in the absence as well as in the presence of PDBu in both types of arteries. The augmentation of contractile responses to caffeine and K+ by phorbol esters in both types of arteries suggests that the phorbols increase the sensitivity of the contractile apparatus to Ca2+, probably by activating protein kinase C. However, the inhibitory effects of phorbols on 5-HT- and AVP-evoked responses in SMA suggest that under these conditions the dominant effect of the phorbols is a marked reduction in the availability of Ca2+ in the SMA but not in the aorta.     

Induction of c-fos and c-myc mRNA by epidermal growth factor or calcium ionophore is cAMP dependent.

Phorbol esters activate protein kinase C and induce expression of the c-fos and c-myc protooncogenes in density-arrested BALB/c 3T3 (A31) cells; in contrast, epidermal growth factor (EGF) does not activate protein kinase C and is a poor inducer of c-fos and c-myc in these confluent cells. We show that, when A31 cells were subconfluent and made quiescent by serum deprivation, the phorbol ester phorbol 12-myristate 13-acetate induced c-fos and c-myc mRNA poorly, whereas EGF was a better inducer. Another platelet-derived growth factor-inducible gene, JE, did not show this differential regulation by phorbol 12-myristate 13-acetate and EGF. The ability of EGF to induce protooncogene mRNA was associated with elevated levels of intracellular cAMP. First, serum-deprived cells maintained cAMP at about 2-fold higher level than density-arrested cells. Second, induction was greatly enhanced by cholera toxin and 3-isobutyl-1-methylxanthine, which increased intracellular cAMP 3- to 10-fold. The calcium ionophore A23187 mimicked EGF in that it elevated c-fos and c-myc mRNA when administered with cholera toxin and isobutylmethylxanthine. Neither cholera toxin and isobutyl-methylxanthine nor A23187 appreciably induced these mRNAs when used alone. Our results suggest that c-fos and c-myc expression can be regulated by an EGF-directed pathway that utilizes calcium and cAMP as cooperating cytoplasmic messengers.     

Analysis of the phorbol ester pharmacophore on protein kinase C as a guide to the rational design of new classes of analogs

The diterpene diester phorbol 12-myristate 13-acetate and the alkaloid teleocidin B are structurally unrelated natural products that display similar potent irritant and tumor-promoting activities. Computer modeling of these and other structural classes of tumor promoters show a marked similarity in the relative positions of certain heteroatoms and hydrophobic groups. For phorbol this mapping consists of the C-4, C-9, and C-20 hydroxyl groups as well as a hydrophobic region filled by a long-chain acyl functionality attached to either the C-12 or the C-13 positions. Diacylglycerols, thought to be the endogenous activators of the major phorbol ester receptor protein kinase C likewise fit this model in a stereospecific fashion. As an initial test of the utility of the model, members of a new and simplified class of activators were synthesized that possess the predicted essential structural features. These compounds all inhibited specific phorbol ester binding to protein kinase C, albeit with low affinity (10-60 microM); further analysis of one derivative, decylhydroxylindole, confirmed that the inhibition of phorbol ester binding was competitive. This same derivative inhibited epidermal growth factor binding in intact Swiss 3T3 cells and studies with another derivative showed phosphorylation of a 40-kDa protein in platelets. Both of these in vivo responses are characteristic of phorbol esters.     

Identification of the proteolytically activated form of protein kinase C in stimulated human neutrophils.

The proteolytically activated form of protein kinase C has been identified in human neutrophils by using a monoclonal antibody that recognizes both the native kinase and the catalytically active proteolytic fragment (protein kinase M). Stimulation with fMet-Leu-Phe results in the conversion of approximately 30% of native protein kinase C to protein kinase M, with little evidence of further degradation. Stimulation with phorbol 12-myristate 13-acetate, on the other hand, causes only a transient formation of protein kinase M, with complete loss of total kinase activity. These differences are related to the differences in biochemical responses, reported earlier, in neutrophils exposed to these two activators.     

Phorbol ester increases the dihydropyridine-sensitive calcium conductance in a vascular smooth muscle cell line

In vascular smooth muscle, phorbol esters cause a slowly developing contraction and an associated transmembrane calcium flux, both of which are inhibited by dihydropyridine calcium channel antagonists. In the A7r5 cultured vascular cell line, we used the whole-cell voltage-clamp technique to identify voltage-dependent calcium conductances and investigate the effect of phorbol esters on that conductance having characteristic dihydropyridine sensitivity (slowly inactivating, high-threshold, "L-type"). With barium as the charge carrier, large-amplitude (100-800 pA) inward currents of two types were characterized by their kinetics and voltage dependence. With holding potential--80 mV, a rapidly inactivating, low-threshold current ("T-type") was activated by depolarizations above-40 mV and was maximal at -10 mV. With holding potential -30 mV, this component was inactivated, and a second slowly inactivating, high-threshold current was activated above -10 mV and was maximal at +10 to +20 mV. These currents are similar to the T-type and L-type currents previously described in vascular smooth muscle cells. When added to the bath, the active phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (100 nM) increased the slowly inactivating (L-type) current by 32 +/- 20% (n = 8, +/- SD). Phorbol-12,13-dibutyrate (100 nM) caused a similar effect, but the inactive phorbol, 4-alpha-phorbol (100 nM), did not. We conclude that at least two distinct calcium conductances are expressed in A7r5 vascular smooth muscle cells, and that the dihydropyridine-sensitive calcium conductance is acutely modulated by phorbol esters, presumably acting through stimulation of protein kinase C. Such modulation may play a role in increasing transmembrane calcium influx mediated by agonist-receptor interactions that lead to activation of protein kinase C and may help to sustain or amplify calcium-dependent cell responses.     

Regulation of a ribosomal protein S6 kinase activity by the Rous sarcoma virus transforming protein, serum, or phorbol ester.

Protein kinase capable of phosphorylating 40S ribosomal protein S6 on serine residues has been detected in chicken embryo fibroblasts. This activity appears to be regulated in direct response to expression of pp60v-src in chicken embryo fibroblasts infected with a temperature-sensitive transformation mutant of Rous sarcoma virus. Partially purified S6 kinase was highly specific for S6 in 40S ribosomal subunits. The S6 kinase was not inhibited by calcium or by the heat-stable inhibitor of cAMP-dependent protein kinase, nor was it activated by phosphatidylserine, diacylglycerol, and calcium. Thus, it is distinct from protein kinase C and cAMP-dependent protein kinase, which are capable of phosphorylating S6 in vitro. The tumor-promoter phorbol 12-myristate 13-acetate also stimulated ribosomal protein S6 kinase activity in serum-starved chicken embryo fibroblasts, whereas phorbol, the inactive analog of phorbol 12-myristate 13-acetate, had no effect. S6 kinase activity stimulated by expression of pp60v-src, by phorbol 12-myristate 13-acetate, or by serum growth factors exhibited similar chromatographic properties upon ion-exchange chromatography. These results suggest that a common protein kinase may be activated by three diverse stimuli all involved in regulating cell proliferation.     

Signal transduction systems in growth hormone-releasing hormone and somatostatin release from perifused rat hypothalamic fragments.

The role of signal transduction systems was examined in the secretion of GH-releasing hormone (GHRH) and somatostatin (SS) from perifused rat hypothalamic fragments. Forskolin, an adenylate cyclase activator, stimulated the release of GHRH and SS in a concentration-dependent manner (10-100 microM) with greatest stimulation for GHRH at 100 microM (mean +/- SE, 249 +/- 14%) and for SS at 30 microM (172 +/- 18%). (Bu)2cAMP also augmented GHRH and SS release. The protein kinase-C activator phorbol 12-myristate 13-acetate did not significantly stimulate basal GHRH or SS release at concentrations of 10 nM to 1 microM. The calcium ionophore A23187 enhanced the release of GHRH and SS in a concentration-dependent manner (2-20 microM), with the greatest responses of 282 +/- 50% at 10 microM and 189 +/- 24% at 20 microM, respectively. Potentiation by phorbol 12-myristate 13-acetate of forskolin-stimulated GHRH and SS release was observed. A23187 at 10 microM did not enhance forskolin-stimulated GHRH release, but did potentiate forskolin-stimulated SS release in a more than additive response. We conclude that there is 1) cAMP stimulation of hypothalamic GHRH and SS release, 2) a modulating role of protein kinase-C on cAMP-stimulated release of GHRH and SS, 3) a stimulatory role of the calcium messenger system for GHRH and SS release, 4) interaction of the signal pathways with differences in net GHRH and SS responses, and 5) a modulatory effect of protein kinase-C in perifused hypothalamic fragments which differs from the stimulation of basal GHRH and SS release reported in fetal-derived hypothalamic cell cultures. Our observations suggest an important regulatory role of interacting signal transduction systems in the hypothalamic secretion of GHRH and SS.     

Sodium and calcium influx induced by phorbol esters in airway smooth muscle cells

The second messenger protein kinase C (PKC) may play a critical role in the regulation of smooth muscle tone. In this study we examined how a direct stimulation of PKC in airway smooth muscle (ASM) cells influences the electrical and contractile properties of these cells. ASM preparations were obtained from adult male guinea pigs (Camm-Hartley strain). Changes in both the resting membrane potential (Em), as measured by a glass microelectrode technique, and the isometric force, measured by a copper-beryllium strain gauge, were continuously monitored. All experiments were done at the optimal length (Lmax) of ASM preparations and a temperature of 37.0 +/- 0.5 degrees C. The effects of phorbol myristate acetate (PMA), phorbol 12,13-diacetate (PDA), and 12,deoxyphorbol-13-isobutyrate (DPB) were investigated (with concentrations ranging from 10(-10) to 10(-4) M). We found that: (1) administration of all three phorbol esters caused a triphasic electrical and contractile response of ASM preparations showing initial, rapid depolarization, an increase in the isometric force, and slow hyperpolarization and relaxation of ASM followed by slow depolarization and an increase in the isometric force; (2) the presence of specific PKC inhibitor H-7 (10(-5) M) prevented development of both electrical and contractile events as induced by tested phorbols; (3) amiloride (10(-5) M) attenuated first- and second-phase responses, whereas furosemide (10(-5) M) inhibited all three responses (a sodium-free environment attenuated all three responses); (4) ouabain (10(-5) M) inhibited a second-phase response, whereas verapamil (10(-6) M) partially attenuated all three phases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol 12-myristate 13-acetate and insulin increase the concentration of fructose 2,6-bisphosphate and stimulate glycolysis in chicken embryo fibroblasts.

Incubation of chicken embryo fibroblasts with mitogenic concentrations of insulin for 24 hr or with the tumor promoter phorbol 12-myristate 13-acetate for 6 hr stimulated lactate release and 3-O-methylglucose uptake. Insulin also increased the Vmax of 6-phosphofructo-1-kinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11). Both agents increased the concentration of fructose 2,6-bisphosphate and the activity of 6-phosphofructo-2-kinase (EC 2.7.1.-), the enzyme that catalyzes the synthesis of this stimulator of 6-phosphofructo-1-kinase. These changes provide an explanation for the stimulation of glycolysis by insulin and phorbol esters. In contrast to the situation in rat liver, fructose 2,6-bisphosphate concentration did not decrease after cyclic AMP treatment. Incubation of cells with phorbol ester analogues or with glycerol derivatives that are known to stimulate, or to bind to, protein kinase C did increase the concentration of fructose 2,6-bisphosphate, suggesting that the stimulation of 6-phosphofructo-2-kinase by phorbol 12-myristate 13-acetate is mediated by protein kinase C.     

Activation of tracheal smooth muscle contraction: synergism between Ca2+ and activators of protein kinase C.

The effects of divalent ionophores (A23187 and ionomycin), Ca2+ channel agonist (BAY K 8644), and protein kinase C (C-kinase) activators [phorbol 12-myristate 13-acetate (PMA), mezerein] on bovine tracheal smooth muscle contraction were investigated. A23187 (5 microM) and ionomycin (0.5 microM) produced a prompt but transient contraction. C-kinase activators either produced no effect--e.g., PMA at 200 nM--or produced a rise in tension that was slow in onset but then gradually increased--e.g., mezerein at 400 nM. In contrast, ionophores and C-kinase activators, in combination, acted synergistically to produce a prompt and sustained contractile response that is reminiscent of that observed in response to carbachol, a cholinergic agonist. In addition, BAY K 8644 (20 nM), which has a minimal effect on tension by itself, could significantly enhance contraction induced by C-kinase activators. The contraction induced by all of these agents was quickly reversed either by removal of extracellular Ca2+ or upon addition of forskolin, an activator of adenylate cyclase. A similar reversal of carbachol-induced contraction by forskolin was observed with carbachol-induced contraction. These findings strongly suggest that C-kinase plays an important role in mediating tracheal smooth muscle contraction.     

Possible involvement of adenylyl cyclase-cAMP-protein kinase a pathway in somatostatin inhibition of growth hormone release from chicken pituitary cells.

Somatostatin (SRIF) reduces growth hormone releasing hormone (GRF)-stimulated growth hormone (GH) release from avian and mammalian adenohypophyseal cells. The present studies examined the intracellular mechanisms mediating SRIF inhibition of GRF-stimulated GH release from chicken pituitary cells. Increases (P less than 0.05) in GH release were observed in the presence of (1) GRF; (2) the adenylyl cyclase stimulator, forskolin; (3) a cAMP analog, 8-bromo-cAMP; (4) the phosphodiesterase inhibitor 3-isobutyl-l-methyl-xanthine (IBMX) combined with GRF; (5) a tumor-promoting phorbol ester and protein kinase C activator, phorbol 12-myristate, 13-acetate (PMA); (6) a diacylglycerol analog, 1,2-dioctanoyl-glycerol (DiC8); and (7) a calcium ionophore, A23187, alone and in combination with PMA. Somatostatin (10 ng/ml) reduced the release of GH stimulated by GRF, forskolin, and 8-bromo cAMP and the GRF-provoked release of GH in the presence of IBMX (P less than 0.05). Somatostatin, however, did not influence GH release in the presence of the protein kinase C activators, PMA or DiC8, or the calcium ionophore A23187. These data suggest that SRIF inhibits GRF-provoked GH release by reducing the ability of the cAMP-protein kinase A but not of the calcium or protein kinase C intracellular message pathways to stimulate GH release.     

Effect of alpha adrenergic agents and phorbol esters on phosphorylation of sarcolemmal proteins in beating guinea pig hearts

STUDY OBJECTIVE--The aim was to determine whether activation of protein kinase C by alpha adrenergic agonists or phorbol esters would be associated with increased phosphorylation of the 15 kDa sarcolemmal protein in guinea pig hearts. DESIGN--Intact, beating guinea pig hearts were perfused with modified Krebs-Henseleit buffer containing [32P]Pi and freeze clamped in a control condition or at the peak of the inotropic response to noradrenaline. Membrane vesicles enriched in sarcolemma were isolated and then subjected to SDS polyacrylamide gel electrophoresis and autoradiography. Phosphorylated proteins were identified and 32P incorporation was quantitated. In some cases, hearts were perfused with phorbol 12-myristate, 13-acetate, or dioctanoyl-glycerol, which are known to be potent activators of protein kinase C. EXPERIMENTAL PREPARATIONS--Whole hearts from 55 anaesthetised guinea pigs weighing 500-600 g were used. MEASUREMENTS AND MAIN RESULTS--Perfusion of guinea pig hearts with noradrenaline resulted in increases in contractility and tissue inositol 1,4,5-triphosphate levels, but there were no increases in the phosphorylation of the 15 kDa sarcolemmal protein observed. Furthermore, perfusion with phorbol 12-myristate, 13-acetate, or dioctanoylglycerol failed to stimulate the phosphorylation of the 15 kDa sarcolemmal protein. CONCLUSIONS--These data indicate that the 15 kDa sarcolemmal protein, which may be phosphorylated by protein kinase C in vitro, is not a substrate for the same enzyme in beating guinea pig hearts.     

Enhanced activation of phospholipase C and insulin secretion from islets incubated in fatty acid-free bovine serum albumin

Incubation in 100 μmol/L fatty acid-free bovine serum albumin (FAF-BSA) significantly amplifies insulin secretion from isolated, perifused rat islets. When compared with the responses of control islets incubated in 100 μmol/L radioimmunoassay-grade BSA, insulin secretion rates were increased 2- to 3-fold when these islets were stimulated with 10 mmol/L glucose alone or with the combination of 10 mmol/L glucose, 15 mmol/L KCl, and 100 μmol/L diazoxide. These amplified secretory responses were paralleled by significant increases in the phospholipase C (PLC) activation monitored by fractional increases in ³H-inositol efflux from these same islets. Amplified PLC responses were also observed with the cholinergic agonist carbachol (50 μmol/L). No differences in the secretory responses to the protein kinase C activator phorbol 12-myristate 13-acetate (200 nmol/L) could be detected between control and FAF-BSA-pretreated rat islets. Mouse islets were also immune to the amplifying impact of this treatment protocol. These findings demonstrate that short-term incubation in FAF-BSA significantly augments the activation of PLC in rat islets by a number of agonists. This proximal event provides the impetus for the distal activation of protein kinase C. If applicable to human islets, this manipulation may provide a mechanism to enhance the secretory responses from islets destined for transplantation, thus improving their in vivo secretory capacity.     

Vascular responsiveness to protein kinase C activators in mineralocorticoid-hypertensive rats

This study characterizes vascular reactivity to protein kinase C activators, 12-O-tetradecanoylphorbol-13-acetate (TPA) and mezerein, in normotensive sham and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Mesenteric arteries were excised, cut helically into strips and placed in a muscle bath for measurement of isometric force generation. Cumulative addition of TPA or mezerein to the bath caused an increase in tension in arteries from hypertensive and normotensive rats. Threshold values for TPA and mezerein (dose that produced a 5 mN/mm2 response) were lower in arteries from DOCA-salt rats (TPA, 0.24 x 10(-8) mol/l; mezerein, 0.32 x 10(-8) mol/l) than in control arteries (TPA, 2.82 x 10(-8) mol/l; mezerein, 2.34 x 10(-8) mol/l). Contractions to TPA in arteries from DOCA hypertensive rats were inhibited by the calcium-channel antagonist verapamil (10(-6) mol/l) to a greater degree than normotensive values. Arteries from rats undergoing DOCA-salt treatment for 5-7 days and from DOCA-treated rats drinking tap water for 4-6 weeks were less responsive to TPA than were arteries from the DOCA-salt hypertensive rats after 4-6 weeks of treatment. Furthermore, responsiveness to TPA in arteries from untreated rats was reduced compared with that in arteries from normotensive rats maintained on high-salt drinking water. Threshold responses to TPA did not differ between arteries incubated with 10(-6) mol/l deoxycorticosterone and those incubated with the vehicle (ethanol). This study demonstrates that arteries from DOCA-salt hypertensive rats are more responsive to the contractile effects of TPA and mezerein than those from normotensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Second messengers involved in the regulation of corticotropin-releasing hormone mRNA and peptide in cultured rat fetal hypothalamic primary cultures

Using primary cultures of dispersed rat fetal hypothalami, we studied the effect of forskolin and the phorbol ester 12-o-tetradecanoyl phorbol 13-acetate, activators of protein kinase A and C, respectively, on corticotropin-releasing hormone (CRH) regulation. CRH mRNA accumulation and peptide release were stimulated by both agents, indicating that the protein kinase A and protein kinase C messenger systems are involved in the regulation of CRH gene expression and are functional in hypothalamic neurons isolated from fetal brain.     

Calcium-stimulated parathyroid hormone-like protein secretion: potentiation through a protein kinase-C pathway

The effect of altering intracellular calcium levels on PTH-like protein (PLP) secretion was assessed in the NCI-H727 cell line. Ionomycin stimulated PLP secretion in a dose- and time-dependent manner, with a maximum rate of secretion occurring within minutes. Phorbol esters (phorbol 12-myristate 13-acetate and phorbol 12,13-didecanoate) stimulated PLP secretion at 10(-7)-10(-5) M, whereas forskolin treatment had no effect. When cells were treated initially with phorbol esters, ionomycin-stimulated PLP secretion was potentiated. The kinetics of this effect were rapid (t1/2, less than 1 min), and pretreatment with cycloheximide or actinomycin-D before phorbol ester and ionomycin treatment did not alter PLP secretion. Pretreatment for 24 h with phorbol 12-myristate 13-acetate to desensitize the cells or use of inactive phorbol ester isomer 4 alpha-phorbol 12,13-didecanoate did not potentiate the ionomycin-stimulated PLP secretion. Treatment with exogenously added phospholipase-C caused a dose-dependent increase in PLP secretion. These data indicate that PLP secretion can be stimulated through a phospholipase-C-mediated mechanism.     

Electrophysiological properties of a cell line of the gonadotrope lineage.

The role of ion channels in the secretion of gonadotropins from anterior pituitary gonadotropes has been difficult to study at the single cell level because the cells are difficult to distinguish from other pituitary cell types. Recently, a cell line, alpha T3-1, has been generated that makes and secretes the alpha-subunit of gonadotropins. These cells have GnRH receptors, but not TRH receptors, and are, thus, specific to the gonadotrope lineage. We have used the patch clamp technique to investigate the types of ion channels expressed in alpha T3-1 cells and to test for electrophysiological responses to GnRH and a phorbol ester. These cells express TTX-sensitive sodium channels with rapid kinetics, several types of potassium channels, including Ca2(+)-sensitive ones, and two types of calcium channels. The currents through calcium channels are augmented by application of 100 nM GnRH or 10 nM phorbol 12-myristate 13-acetate, a phorbol ester. The augmentation by GnRH and phorbol 12-myristate 13-acetate is consistent with other reports that a portion of stimulated gonadotropin release is dependent on external calcium and sensitive to block by dihydropyridine antagonists. Thus, this cell line may be useful for studies of mechanisms underlying responses to GnRH.