Binding of [3H]bryostatin 4 to protein kinase C.

The bryostatins represent a unique class of activators of protein kinase C (PKC) which induce only a subset of the responses typical of the phorbol esters and block those responses to the phorbol esters which they themselves do not induce. To better understand the interaction of the bryostatins with PKC, we have synthesized [26-3H]bryostatin 4 and characterized its binding to PKC. [3H]Bryostatin 4 and [3H]phorbol 12,13-dibutyrate ([3H]PDBu) differed markedly in their binding to PKC reconstituted with phosphatidylserine (PS). The binding affinity of [3H]bryostatin 4 under these conditions was too high to measure and the rate of release of bound bryostatin was much slower than that of the phorbol esters, with a half-time of several hours. These properties caused bryostatin 1 to appear to inhibit [3H]PDBu binding under these conditions in a non-competitive fashion. Both the high potency and the slow rate of release of the bryostatins may contribute to their unique pattern of biological activity. By reconstituting PKC in a mixture of 1.5% Triton X-100:0.3% PS, we were able to establish reversible conditions for [3H]bryostatin 4 binding. Under these latter conditions, binding of [3H]bryostatin 4 was competitively inhibited by PDBu, consistent with both the bryostatin and phorbol esters binding to PKC in a qualitatively similar fashion. Binding affinities to PKC isozymes alpha, beta, and gamma were compared and little difference was found, suggesting that differential recognition by these isozymes does not account for the unique biological activity of the bryostatins.     

Role of protein kinase C in desensitization of spinal delta-opioid-mediated antinociception in the mouse.

1. Receptor phosphorylation and down-regulation by protein kinases may be a key event initiating desensitization. The present studies were designed to investigate the effect of a potent protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), on antinociception induced by intrathecal (i.t.) administration of a selective delta-opioid receptor agonist [D-Ala2] deltorphin II in the male ICR mouse and on the specific binding of [3H]-[D-Ser2, Leu5]enkephalin-Thr6 (DSLET), a delta-opioid receptor ligand, in the crude synaptic membrane of the spinal cord. 2. Intrathecal (i.t.) pretreatment with PDBu at low doses, which injected alone did not affect the basal tail-flick latency, dose-dependently attenuated the antinociception induced by i.t. administration of [D-Ala2]deltorphin II. The attenuation of i.t.-administered [D-Ala2] deltorphin II-induced antinociception by PDBu was reversed in a dose-dependent manner by i.t. concomitant pretreatment with a specific PKC inhibitor, calphostin C. 3. In the binding experiment, incubation of the crude synaptic membrane of the spinal cord for 2 h at 25 degrees C with PDBu (0.03 to 10 microM) caused a dose-dependent inhibition of the [3H]-DSLET binding. Scatchard analysis of [3H]-DSLET binding revealed that PDBu at 10 microM displayed a 30.7% reduction in the number of [3H]-DSLET binding sites with no significant change in affinity, compared with the non-treatment control, indicating that the activation of membrane-bound PKC by PDBu causes a decrease in the number of specific delta-opioid agonist binding sites. 4. An i.t. injection of [D-Ala2]deltorphin II produced an acute antinociceptive tolerance to the antinociceptive effect of a subsequent i.t. challenge of [D-Ala2]deltorphin II. Concomitant pretreatment with calphostin C markedly prevented the development of acute tolerance to the i.t.-administered [D-Ala2]deltorphin II-induced antinociception. On the other hand, a highly selective protein kinase A (PKA) inhibitor, KT5720, did not have any effect on the development of acute tolerance to [D-Ala2]deltorphin II antinociception. 5. These findings suggest that a loss of specific delta-agonist binding by the activation of PKC by PDBu is involved in the PDBu-induced antinociceptive unresponsiveness to delta-opioid receptor agonist in the mouse spinal cord. Based on the acute tolerance studies, we propose that PKC, but not PKA, plays an important role in the process of homologous desensitization of the spinal delta-opioid receptor-mediated antinociception.     

Distribution of protein kinase C in the hippocampus of the gerbil and rat: autoradiographic analysis by [3H]phorbol 12,13-dibutyrate.

In-vitro quantitative receptor autoradiography with [3H]phorbol 12,13-dibutyrate (PDBu) was used to determine the affinity constant (Kd) and the maximum number of receptor sites (Bmax) for protein kinase C (PKC) in subregions of the gerbil hippocampus, and to compare the distribution of [3H]PDBu binding sites in the gerbil hippocampus with that in the rat hippocampus. The Kd and Bmax values in the subregions of the gerbil hippocampus were estimated at 2.6-3.8 nM and 2.38-2.54 pmol (mg tissue)-1, respectively. The distribution of hippocampus [3H]PDBu binding sites was uniform in the gerbil but not in the rat. The [3H]PDBu binding activities in the strata oriens of the CA1 and CA3 subfields and the molecular layer of the dentate gyrus in the rat hippocampus were significantly higher than in the gerbil hippocampus. However, binding activity in the stratum lacunosum-moleculare of the rat CA1 subfield was statistically lower. These data demonstrate a difference in the distribution of [3H]PDBu binding activity in the hippocampus between the gerbil and rat.     

Regional and temporal profiles of phorbol 12,13-dibutyrate binding after myocardial infarction in rats: effects of captopril treatment

Phosphoinositide turnover and protein kinase C (PKC) mediate the signaling of angiotensin II, which plays a pivotal role in ventricular remodeling after myocardial infarction (MI). To determine whether PKC is activated after MI, rat hearts after MI were subjected to in vitro quantitative autoradiography with [3H]phorbol 12,13-dibutyrate (PDBu), which is highly selective for PKC. [3H]PDBu binding in the infarcted area increased significantly compared with the non-infarcted region 7 and 21 days after MI, but not 1 and 3 days and 10 months after MI. [3H]PDBu binding in the noninfarcted area was similar to that in the sham-operated rats. Immunohistochemical analysis revealed that abundant macrophages (7 days after MI), fibroblasts, and myofibroblasts (7 and 21 days after MI) occupied the infarcted region. To investigate whether myocardial [3H]PDBu binding is affected by captopril, hearts were subjected to in vitro autoradiography with [3H]PDBu after 1- or 3-week captopril treatment or no treatment. Captopril treatment significantly suppressed [3H]PDBu binding in the infarcted area 3 weeks after MI, but not 1 week after MI nor in the noninfarcted areas. These results suggest that PKC is upregulated during the healing and fibrogenic process after MI and that captopril treatment suppresses the upregulation in the infarcted area.     

Homologous histamine H1 receptor desensitization results in reduction of H1 receptor agonist efficacy.

Prolonged exposure of the guinea-pig intestinal longitudinal smooth muscle to histamine caused homologous desensitization of the H1 receptor, which led to reduced H1 receptor-mediated production of [3H]inositol phosphates as well as to reduced H1 agonist-induced contractions. [3H]Mepyramine binding studies showed that desensitization affected neither the agonist affinity nor the number of H1 receptors. Combining the data from the binding studies and the contraction measurements it was found that desensitization results in a selective reduction of agonist efficacy.     

c-Jun N-terminal kinase contributes to norepinephrine-induced contraction through phosphorylation of caldesmon in rat aortic smooth muscle

Vascular smooth muscle contraction is mediated by activation of extracellular signal-regulated kinase (ERK) 1/2, an isoform of mitogen-activated protein kinase (MAPK). However, the role of stress-activated protein kinase/c-Jun N-terminal kinase (JNK) in vascular smooth muscle contraction has not been defined. We investigated the role of JNK in the contractile response to norepinephrine (NE) in rat aortic smooth muscle. NE evoked contraction in a dose-dependent manner, and this effect was inhibited by the JNK inhibitor SP600125. NE increased the phosphorylation of JNK, which was greater in aortic smooth muscle from hypertensive rats than from normotensive rats. NE-induced JNK phosphorylation was significantly inhibited by SP600125 and the conventional-type PKC (cPKC) inhibitor G?6976, but not by the Rho kinase inhibitor Y27632 or the phosphatidylinositol 3-kinase inhibitor LY294002. Thymeleatoxin, a selective activator of cPKC, increased JNK phosphorylation, which was inhibited by G?6976. SP600125 attenuated the phosphorylation of caldesmon, an actin-binding protein whose phosphorylation is increased by NE. These results show that JNK contributes to NE-mediated contraction through phosphorylation of caldesmon in rat aortic smooth muscle, and that this effect is regulated by the PKC pathway, especially cPKC.     

Preconditioning of coronary artery against vasoconstriction by endothelin-1 and prostaglandin F2alpha during repeated downregulation of epsilon-protein kinase C

The cellular mechanisms of coronary vasospasm are unclear, and a role for protein kinase C (PKC) activation by the endogenous vasoconstrictors endothelin-1 (ET-1) and prostaglandin F2alpha (PGF2alpha) has been suggested. In this study, we developed a phorbol ester-induced PKC downregulation protocol to investigate the relation between the amount and activity of specific PKC isoforms in coronary arterial smooth muscle and coronary vasoconstriction by ET-1 and PGF2alpha. Isometric tension was measured in deendothelialized porcine coronary artery strips, [Ca2+]i was monitored in single coronary smooth muscle cells loaded with fura-2, and the whole tissue, cytosolic, and particulate fractions were examined for PKC activity and reactivity with isoform-specific anti-PKC antibodies using Western blot analysis. In Ca(2+)-free (2 mM EGTA) Krebs solution, ET-1 (10(-7) M), PGF2alpha (10(-5) M) and PKC activator phorbol 12,13-dibutyrate (PDBu) (10(-6) M) caused significant contractions that were completely inhibited by the PKC inhibitors staurosporine and calphostin C, no significant change in [Ca2+]i, and significant activation and translocation of the Ca(2+)-independent epsilon-PKC but not the Ca(2+)-dependent alpha-PKC. In Ca(2+)-free Krebs, a single application of PDBu produced maximal contraction and PKC activity after 30 min, which declined to basal levels in 3 h and remained steady for 24 h, but did not prevent subsequent increases in contraction and PKC activity with a new addition of PDBu and did not significantly decrease the amount of alpha- or epsilon-PKC. Repeated (five to eight) applications of PDBu in Ca(2+)-free Krebs at 3-h intervals completely inhibited subsequent increases in contraction and PKC activity to PDBu, ET-1, or PGF2alpha, and significantly decreased the amount of epsilon-PKC but not that of alpha-PKC. These results provide evidence that a Ca(2+)-independent coronary vasoconstriction induced by ET-1 and PGF2alpha is associated with activation of the epsilon-PKC isoform. The results suggest that, in coronary artery smooth muscle, downregulation of PKC is isoform specific and is more dependent on the frequency rather than the duration of PKC activation. The results also suggest that repeated downregulation of epsilon-PKC might play a role in preconditioning of the coronary artery against vasoconstriction by ET-1 and PGF2alpha.     

Characterization of specific [3H]dimethylstaurosporine binding to protein kinase C

The microbial alkaloid staurosporine is a member of a recently described family of protein kinase inhibitors. [N,N-dimethyl-3H]N-dimethylstaurosporine ([3H]DMS) was prepared from staurosporine by methylation with [3H]methyl iodide. Since staurosporine inhibits protein kinase C (PKC) most potently, the binding of [3H]DMS to this enzyme was examined. Unlike [20-3H(N)]phorbol-12,13-dibutyrate ([3H]PDBu) binding to PKC, [3H]DMS binding was not calcium or phosphatidylserine (PS) dependent. Binding was reversible, with a T1/2 of 69 min and a Koff of 0.01/min. Non-specific binding was defined by a 500-fold molar excess of staurosporine and was less than 10% of total [3H]DMS binding. Specific binding of [3H]DMS was consistent with a single class of binding sites with a Kd of 3.8 +/- 0.6 nM and a Bmax of 675 +/- 30 pmol/g tissue. In competition experiments, staurosporine inhibited [3H]DMS binding with a Ki of 4.7 +/- 0.6 nM, indicating that the two alkaloids had a similar potency for PKC. Also, unlabeled DMS and staurosporine inhibited [3H]DMS binding and PKC catalysis with equivalent potencies. Highly purified rat brain PKC bound equimolar amounts of [3H]PDBu and [3H]DMS. In contrast, crude rat brain PKC, which had been proteolysed to generate a PS and Ca2+ independent enzyme (PK-M) retained the ability to bind [3H]DMS, but not [3H]PDBu. In addition, the kinase inhibitors K-252a and H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine] inhibited [3H]DMS binding, whereas PDBu did not. These results indicate that [3H]DMS is a useful ligand to identify catalytic inhibitors of kinase activity and to explore their mechanisms of action.     

The guanine nucleotide exchange factor RasGRP is a high -affinity target for diacylglycerol and phorbol esters

RasGRP is a recently described guanine nucleotide exchange factor (GEF) that possesses a single C1 domain homologous to that of protein kinase C (PKC). The phorbol ester [(3)H]phorbol 12, 13-dibutyrate ([(3)H]PDBu) bound to this C1 domain (C1-RasGRP) with a dissociation constant of 0.58 +/- 0.08 nM, similar to that observed previously for PKC. Likewise, the potent PKC activator bryostatin 1, a compound currently in clinical trials, showed high affinity binding for C1-RasGRP. Structure activity analysis using several phorbol ester analogs showed both similarities and differences in ligand selectivity compared with PKC; the differences were comparable in magnitude to those between different PKC isoforms. Similarly, the potency of the PKC inhibitor calphostin C to inhibit [(3)H]PDBu binding to C1-RasGRP was similar to that observed for PKC. In contrast to the relative similarities in ligand recognition, the lipid cofactor requirements differed between RasGRP and PKC. The C1 domain plus the EF-hand motif of RasGRP (C1EF-RasGRP) was markedly less dependent on acidic phospholipids than was PKCalpha. The differences in lipid requirements were reflected in differential ligand selectivity under conditions of limiting lipid. Despite the presence of twin EF-hand like motifs, calcium did not affect the binding of [(3)H]PDBu to C1EF-RasGRP. We conclude that RasGRP is a high affinity receptor for phorbol esters and diacylglycerol. RasGRP thus provides a direct link between diacylglycerol generation or phorbol ester/bryostatin treatment and Ras activation.     

A selective effect of protein kinase C activators on noradrenaline release compared with subsequent contraction in canine isolated saphenous veins.

1. Effects of protein kinase C (PKC) activators and inhibitors on both tritium overflow and subsequent contraction evoked by transmural nerve stimulation (TNS) were investigated in canine saphenous veins prelabelled with [3H]-noradrenaline. 2. Activation of PKC by stepwise increasing concentrations (0.01 nM-1 microM) of 12-O-tetradecanoylphorbol 13-acetate (TPA), phorbol 12,13-dibutyrate (PDBu) or mezerein caused a significant and concentration-dependent enhancement of the tritium overflow evoked by TNS, while the activators failed to affect the corresponding contraction except with the highest concentration of PDBu when the contraction was significantly reduced. Phorbol, which is inactive on PKC, had no effects on the tritium overflow and contraction induced by TNS. 3. PKC inhibitors, polymyxin B (1 and 10 microM) and the isoquinolinesulphonamide, H-7 (1 microM), inhibited significantly the phorbol ester-potentiated tritium overflow evoked by TNS with no effects on the contraction. H-7 and the related inhibitor H-8 at 10 microM reduced significantly both responses to TNS in the presence of TPA, while they suppressed only the TNS-induced contraction in the absence of TPA. 4. None of the PKC activators or inhibitors affected the spontaneous tritium overflow. 5. PDBu (0.01 and 0.1 microM) elevated resting tension of the veins more effectively than TPA and mezerein. 6. These results suggest that PKC may modulate electrically stimulated noradrenaline release from adrenergic nerve endings of the canine saphenous veins and the PKC activators may act more selectively on presynaptic than postsynaptic sites, but have no apparent effect on postjunctional noradrenergic mechanisms.     

Histaminergic H1-receptors in smooth muscle and endothelium of bovine thoracic aorta

The vascular endothelium modulates relaxation and contraction of blood vessels. Since endothelial cells respond to a variety of vasoactive substances, it was suggested that specific cell membrane receptors exist on the endothelial cells which are responsible for the modulatory role of the endothelium on the blood vessels. We therefore investigated the localization and binding characteristics of histaminergic H1-receptors in the vascular model system of the bovine thoracic aorta. Our earlier binding experiments showed that histaminergic H1-receptor binding sites labelled with [3H]mepyramine are present on the vascular smooth muscle membranes of this tissue. In addition a small number of specific H1-receptor binding sites also exist on the endothelial cells of this tissue with the following binding characteristics: Bmax = 34.6 fmol [3H]mepyramine/mg protein, KD = 2.13 nM. [3H]mepyramine binding is more effectively inhibited by H1- than H2-receptor agonists and antagonists. These results provide evidence for the existence of endothelial histaminergic H1-receptor binding sites in addition to vascular smooth muscle H1-receptors in the bovine thoracic aorta.     

Synergism between phorbol ester and the Ca2+ ionophore A23187 on protein kinase C translocation, [3H]PDBu binding and adenosine A2-receptor activation in Jurkat cells

Phorbol 12,13-dibutyrate (PDBu) enhances 5'-(N-ethylcarboxamido)-adenosine (NECA) stimulated cyclic adenosine 3',5'-monophosphate (cAMP) production in the human T-cell leukemia line, Jurkat. Addition of the Ca2+ ionophore A23187 lowered the EC50 value for PDBu from 49 to 7.1 nM. In binding experiments, where intact cells were incubated with [3H]PDBu at 37 degrees C, addition of A23187 increased the number of binding sites for the phorbol ester. Pretreatment of cells with A23187 was not sufficient to increase [3H]PDBu binding; A23187 had to be combined with phorbol ester in order to enhance [3H]PDBu binding. PDBu treatment translocated protein kinase from the cytosol to the membrane. This effect of the phorbol ester could be enhanced with A23187 whereas A23187 per se had no effect on protein kinase C distribution. From these data it is concluded that the synergism between A23187 and PDBu, monitored as enhancement of NECA-stimulated cAMP accumulation and increase in [3H]PDBu binding, is paralleled by translocation of the enzyme to the particulate fraction of the cells. The finding that cells where the cellular content of protein kinase C had been translocated to the membrane compartment bound more [3H]PDBu than control cells also suggests that [3H]PDBu binding to intact cells reflects the amount of membrane bound-, rather than the total cellular-enzyme content.     

Homologous and heterologous desensitization of histamine H1- and ATP-receptors in the smooth muscle cell line, DDT1MF-2: the role of protein kinase C.

1. The possible role of protein kinase C (PKC) in homologous and heterologous desensitization of histamine H1- and ATP-receptors has been studied in monolayers of cultured vas deferens smooth muscle cells (DDT1MF-2). Cells were loaded with the calcium-sensitive fluorescent dye fura-2 and increases in intracellular free Ca2+ concentration ([Ca2+]i) monitored in response to histamine H1- or ATP-receptor activation. 2. Histamine and ATP stimulated the release of Ca2+ from intracellular Ca2+ stores and Ca2+ influx across the plasma membrane. Activation of PKC with the phorbol ester beta-phorbol-12,13 dibutyrate (PDBu; 1 microM) attenuated histamine (100 microM) and ATP (10 microM)-induced release of intracellular Ca2+ and Ca2+ influx. 3. The selective PKC inhibitor, Ro 31-8220 (10 microM), reversed the PDBu-induced attenuation of histamine (100 microM)-stimulated Ca2+ responses. 4. Histamine H1- and ATP-receptors are readily susceptible to homologous desensitization since short-term exposure to histamine or ATP (450 s) attenuated the Ca2+ responses elicited by a second application of the same agonist. Furthermore, H1-receptor activation-induced heterologous desensitization of ATP stimulated Ca2+ responses and vice versa. 5. Homologous and heterologous desensitization of histamine and ATP Ca2+ responses still occurred in the presence of the PKC inhibitor, Ro 31-8220 (10 microM). 6. These data suggest that PKC activation can attenuate histamine H1- and ATP-receptor mediated Ca2+ responses. However, based on our experimental data, PKC-independent mechanisms appear to be involved in the homologous and heterologous desensitization of histamine H1- and ATP-receptor mediated Ca2+ responses in DDT1MF-2 cells.     

Protein kinase C-mediated intracellular alkalinization in rat and rabbit aortic smooth muscle cells

The influence of protein kinase C (C-kinase) activation on intracellular pH (pHi) of cultured rat (RASM) and rabbit (RBASM) aortic smooth muscle cells was studied by employing a pH-sensitive fluorescent-dye 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The known C-kinase activators 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol 12,13-dibutyrate (PDBu) and mezerine as well as the agonist angiotensin II each caused an intracellular alkalinization of approximately 0.1-0.15 pH units in RASM and RBASM cells grown in serum-free conditions. TPA-induced alkalinization was sensitive to the Na+/H+ exchange blockers amiloride and 5-N-ethylisopropyl-amiloride (EIPA). These results suggest that protein kinase C activation leads to intracellular alkalinization in vascular smooth muscle cells and the increase in pHi might play an important role in receptor-coupled arterial contraction.     

Vascular protein kinase C in Wistar-Kyoto and spontaneously hypertensive rats.

Phorbol esters which activate protein kinase C (PKC) produced concentration-related force development in aorta from spontaneously hypertensive rat (SHR) and Wistar-Kyoto rat (WKY); all were 2-7 x more potent in SHR. However, total PKC activity in aortas, as well as carotid, caudal and renal arteries, was not different, when SHR was compared with WKY. Binding of phorbol dibutyrate to particulate aortic PKC was similar in SHR and WKY (same apparent Kd and Bmax values), as was potency for displacement of phorbol dibutyrate by phorbol myristate acetate. Furthermore, there was no difference in potency with staurosporine, H-7, and calmidazolium in inhibiting SHR and WKY aortic PKC. These data demonstrate enhanced contractile sensitivity to PKC-activating phorbol esters in SHR aortic smooth muscle that is not related to activity, phorbol ester binding, or sensitivity to inhibitors when SHR PKC is compared with WKY PKC. Thus, signal transduction events distal to PKC activation may be responsible for enhanced vascular contractile sensitivity to phorbol esters in SHR.     

Demonstration of sub-nanomolar affinity of bryostatin 1 for the phorbol ester receptor in rat brain

The effect of bryostatin 1 on [26-3H]phorbol 12,13-dibutyrate [( 3H]PDBu) binding to a washed particulate preparation from rat brain was examined. Bryostatin 1 inhibited phorbol ester binding at concentrations considerably lower than previously reported. As would be expected for a ligand of high affinity, the apparent displacing potency of bryostatin 1 was dependent on the concentration of tissue/binding sites included in the assay. Decreasing the concentration of [3H]PDBu binding sites to the picomolar detection limit resulted in apparent bryostatin displacing potencies in the picomolar range with these values representing an upper estimate of the true affinity. When included in saturation studies with [3H]PDBu, bryostatin 1 displayed mixed competitive/non-competitive inhibition. Using either repetitive washing or dialysis of the membrane preparation, it was not possible to reverse the inhibition produced by bryostatin 1. The greater affinity of bryostatin 1 compared to other classes of agents that act directly on protein kinase C and the stability of its association may contribute to the unique biological properties of the bryostatins.     

Effects of phorbol ester on carbachol-induced contraction in bovine ciliary muscle: possible involvement of protein kinase C

The aim of the research was to characterize muscarinic receptors of bovine ciliary muscle and to investigate the desensitization process. The role of protein kinase C was analyzed. The results show that muscarinic receptors of bovine ciliary muscle have the pharmacological characteristics of the M₃ subtype. Acute exposure to phorbol esters (1 μM phorbol 12,13-dibutyrate, PDB, or 0.1 μM phorbol 12-myristate 13-acetate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensitization and loss of phorbol ester-induced desensitization. Staurosporine (3 μM) and H₇ [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 μM), protein kinase C inhibitors, produced a significant potentiation of the contractile effect of carbachol, reduced the desensitization produced by repeated addition of carbachol and suppressed that induced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [³H]quinuclidinyl benzilate. In vitro incubation with PDB and PMA produced, as expected, a significant translocation of protein kinase C from the cytosol to the membrane. The incubation of the ciliary muscle with carbachol, using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ciliary muscle is correlated, at least partially, to the translocation of protein kinase C from the cytosol to the membrane. The desensitization by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the density and affinity of muscarinic receptors is not involved.     

Desensitization of alpha-adrenergic receptor-mediated smooth muscle contraction: role of the endothelium.

Desensitization of alpha-adrenergic receptor-mediated smooth muscle contraction occur in aortas from New England Deaconess Hospital (NEDH) rats harboring pheochromocytoma (PHEO) and following chronic exposure to the alpha-adrenergic agonist phenylephrine in vitro. Endothelium is known to release an endothelial cell-derived relaxing factor that promotes smooth muscle relaxation. We wondered if the endothelium might contribute to the desensitization of contraction. The role of the endothelium in desensitization was studied using aortic rings with endothelium [E(+)] and with endothelium removed [E(-)]. Maximal phenylephrine (PE)-induced contraction (Emax) for E(+) was 1.7 +/- 0.3 g in controls and 0.4 +/- 0.1 g in PHEO (p less than 0.001), demonstrating desensitization; however, for E(-), Emax was 2.4 +/- 0.2 g in PHEO vs. 2.5 +/- 0.2 g in controls, demonstrating restoration of maximal contraction when the endothelium was removed. However, sensitivity [-log EC50(M)] to PE in E(-) remained significantly lower in PHEO compared to controls (6.94 +/- 0.12 vs. 7.51 +/- 0.14, respectively, p less than 0.001). Similarly, in aortic ring segments desensitized in vitro with phenylephrine, the maximal contraction in phenylephrine-exposed aortas was 60% of that seen in controls. Removal of the endothelium from the vessels pretreated with phenylephrine fully restored the maximal response and sensitivity of these vessels. Treatment of desensitized vessels with hemoglobin (5 x 10(-5) M) restored the maximal contraction and sensitivity to phenylephrine. When the endothelium was removed prior to chronic exposure to phenylephrine, the sensitivity to phenylephrine decreased while the Emax remained similar to controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of high affinity binding of calcium channel blocking drugs to vascular smooth muscle and cardiac sarcolemmal membranes

The binding of the 1,4-dihydropyridine calcium channel blocker [3H]nitrendipine to canine cardiac sarcolemmal and bovine aortic membranes was found to be rapid, specific, saturable, and reversible. Dissociation constants (Kd) determined by Scatchard analysis were 0.14 and 0.16 nM and the maximal numbers of binding sites (Bmax) were 0.96 +/- 0.2 and 0.08 +/- 0.01 pmole/mg protein for cardiac and aortic membranes respectively. Values of Kd calculated from kinetic data were approximately 0.10 nM for both membrane preparations. Competition assays with the enantiomers of a nisoldipine derivative indicated that [3H]nitrendipine binds stereoselectively. The order of potency of several nifedipine analogs for inhibition of binding of [3H]nitrendipine to cardiac and aortic membranes paralleled their relative potencies for inhibition of contraction in smooth muscle. It is concluded that the high affinity binding sites for nitrendipine in bovine aortic smooth muscle membranes are similar to those of canine ventricular sarcolemma.     

Ligand structure-activity requirements and phospholipid dependence for the binding of phorbol esters to protein kinase D

Although protein kinase D (PKD), like protein kinase C (PKC), possesses a C1 domain that binds phorbol esters and diacylglycerol, the structural differences from PKC within this and other domains of PKD imply differential regulation by lipids and ligands. We characterized the phorbol ester and phospholipid binding properties of a glutathione S-transferase-tagged full-length PKD and compared them with those of PKC-alpha and -delta. We found that PKD is a high-affinity phorbol ester receptor for a range of structurally and functionally divergent phorbol esters and analogs and showed both similarities and differences in structure-activity relations compared with the PKCs examined. In particular, PKD had lower affinity than PKC for certain diacylglycerol analogs, which might be caused by a lysine residue at the 22 position of the PKD-C1b domain in place of the tryptophan residue at this position conserved in the PKCs. The membrane-targeting domains in PKD are largely different from those in PKC; among these differences, PKD contains a pleckstrin homology (PH) domain that is absent in PKC. However, phosphatidylinositol-4,5-bisphosphate PIP2, a lipid ligand for some PH domains, reconstitutes phorbol 12,13-dibutyrate (PDBu) binding to PKD similarly as it does to PKC-alpha and -delta, implying that the PH domain in PKD may not preferentially interact with PIP2. Overall, the requirement of anionic phospholipids for the reconstitution of [3H]PDBu binding to PKD was intermediate between those of PKC-alpha and -delta. We conclude that PKD is a high-affinity phorbol ester receptor; its lipid requirements for ligand binding are approximately comparable with those of PKC but may be differentially regulated in cells through the binding of diacylglycerol to the C1 domain.