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Phorbol ester tumor promoters activate protein kinase C (PKC) isozymes and novel non-kinase receptors, suggesting a high degree of complexity in the signaling mechanisms of tumorigenesis. Many studies have shown that PKC isozymes contribute to the progression of malignant phenotype. We review the emerging understanding of the roles of PKC isozymes in the three sequential cellular processes of tumor invasion and metastasis: attachment to extracellular matrix or basement membrane components, matrix degradation by proteolytic enzymes, and migration through the digested matrix. In addition, we discuss the potential role of chimaerins, novel non-kinase phorbol ester receptors, in carcinogenesis.

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Phorbol ester binding was studied in protein kinase C-containing extracts obtained from Trypanosoma cruzi epimastigote forms. Specific 12-O-tetradecanoyl phorbol 13-acetate, [3H]PMA, or 12,13-O-dibutyryl phorbol, [3H]PDBu, binding activities, determined in T. cruzi epimastigote membranes, were dependent on ester concentration with a Kd of 9x10(-8) M and 11.3x10(-8) M, respectively. The soluble form of T. cruzi protein kinase C was purified through DEAE-cellulose chromatography. Both protein kinase C and phorbol ester binding activities co-eluted in a single peak. The DEAE-cellulose fraction was further purified into three subtypes by hydroxylapatite chromatography. These kinase activity peaks were dependent on Ca2+ and phospholipids and eluted at 40 mM (PKC I), 90 mM (PKC II) and 150 mM (PKC III) phosphate buffer, respectively. Western blot analysis of the DEAE-cellulose fractions, using antibodies against different isoforms of mammalian protein kinase C enzymes, revealed that the parasite expresses high levels of the alpha-PKC isoform. Immunoaffinity purified T. cruzi protein kinase C, isolated with an anti-protein kinase C antibody-sepharose column, were subjected to phosphorylation in the absence of exogenous phosphate acceptor. A phosphorylated 80 kDa band was observed in the presence of Ca2+, phosphatidylserine and diacylglycerol.

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1. The relaxant response and cAMP production mediated by stimulation of isoproterenol is reduced in uterine rings from clenbuterol treated rats (0.25 mg kg-1 s.c. 24 hr before experiments) precontracted with 50 mM KCl. 2. Forskolin, in contrast, showed similar relaxant responses in untreated or clenbuterol treated rats. 3. Isoproterenol produces a biphasic response that is composed of a rapid relaxation followed by a slower regaining of tension, which is considered as desensitization. 4. The kinetic study demonstrates marked changes in the desensitization process of beta-adrenoceptors after clenbuterol administration.

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The possible role of cyclic AMP (cAMP) on tritium overflow evoked by stimulation of the cardio-accelerant nerves was studied in rat atria preincubated with [3H]-noradrenaline. Addition of the activator of adenylate cyclase forskolin (1 mumol/l), or of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 mumol/l), did not affect both basal and evoked overflow. However, in the presence of the alpha 2-adrenoceptor antagonist yohimbine (0.03 mumol/l) both forskolin and IBMX increased the stimulation-induced transmitter overflow by 49% and 141%, respectively (compared to yohimbine 0.03 mumol/l). Thus, in rat atria the cAMP-dependent facilitation of noradrenaline release is only present when the autoinhibition exerted by activation of prejunctional alpha 2-adrenoceptors is blocked. Propranolol (0.1 mumol/l) that did not produce any effect on noradrenaline release markedly reduced the facilitatory response induced by forskolin in the presence of yohimbine. When rats were pretreated with the beta 2-adrenoceptor agonist clenbuterol (0.3 mg.kg-1, s.c., twice daily, 14 days), a treatment which desensitizes beta-adrenoceptor-mediated facilitation of noradrenaline release (Kazanietz and Enero 1989), the facilitatory effect of forskolin and IBMX in the presence of yohimbine was abolished. The results indicate that in rat atria the effect of forskolin and IBMX on noradrenaline release are only to be observed after blockade of presynaptic alpha 2-adrenoceptor autoinhibition. beta-adrenoceptor blockade or clenbuterol pretreatment decreases the facilitatory response to forskolin and hence prejunctional beta-adrenoceptor-mediated enhancement of noradrenaline release is linked to the stimulation of adenylate cyclase.

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KCl-contracted aortic rings from 18-month-old rats, in contrast with those from 2-month-old rats, showed a substantial reduction in the relaxant effects of the non-selective beta-adrenoceptor agonist, isoproterenol, and of the selective beta 2-adrenoceptor agonist, clenbuterol, without changes in the relaxant actions of forskolin (an activator of the adenylate cyclase), 3-isobutyl-1-methyl-xanthine (a phosphodiesterase inhibitor) or acetylcholine (an endothelium- and cyclic GMP-dependent vasodilator). The relaxant responses induced by adenosine and 2-Cl-adenosine were also reduced in aged aortas. Isoproterenol and cholera toxin (an inhibitor of GTPase activity of the stimulatory GTP-binding protein) reduced cAMP production in aortas from 18-month-old rats. It is suggested that a decrease in the function of the stimulatory GTP-binding protein may contribute at least in part to the impairment in the vasodilation induced by activation of beta-adrenoceptors in aortas from aged rats.

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Seven or 14 days of treatment with the beta 2-adrenoceptor agonist clenbuterol, 0.3 mg kg-1, s.c., twice daily, increased the basal mean blood pressure in normotensive urethane-anaesthetized rats. The elevated pressure values were maintained until 48 h after the end of the 14 day treatment. Clenbuterol treatment decreased the vasodilatory responses to the beta-adrenoceptor agonist isoprenaline and adenosine, agents which act through an increase in intracellular cyclic AMP. Decreased responses were maintained until 48 h after a 14 day treatment with clenbuterol. On the other hand, its administration to rats for 14 days did not modify the vasodilator responses to acetylcholine or sodium nitroprusside, two agents that exert their effects by enhancing cyclic GMP. The increase in mean blood pressure in urethane-anaesthetized rats after clenbuterol treatment may be a consequence of a reduced vasodilator beta 2-adrenoceptor-mediated response to circulating catecholamines.

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K(+)-contracted rat uterine rings were relaxed in a concentration-dependent manner by the benzodiazepines Ro 5-4864, diazepam and clonazepam, as well as by the putative peripheral benzodiazepine antagonist PK 11195. The relaxation induced by diazepam was not counteracted by the central antagonist Ro 15-1788 (10 microM), and the relaxant effects of Ro 5-4864 and of diazepam were not prevented by either the GABAA antagonist bicuculline (10 microM) or the beta-adrenoceptor antagonist propranolol (1 microM). The mechanism underlying the relaxant effects of benzodiazepines on K(+)-contracted uterine rings is still under study.

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Administration to rats of the selective beta-2 adrenoceptor agonist (+/-)-clenbuterol (CLEN) (0.3 mg.kg-1 s.c., twice daily for 14 days) decreased the relaxant responses to the beta adrenoceptor agonist (-)-isoproterenol (IS) and to CLEN in KCl-contracted aortic rings. The treatment did not modify the vasodilation induced by forskolin (a direct activator of the catalytic subunit of the adenylate cyclase), 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor), adenosine or acetylcholine. IS increased (cAMP) cyclic AMP levels dose-dependently in rat aorta, and this effect was reduced markedly in arteries from CLEN-treated rats. By contrast, the treatment did not modify the forskolin-induced cAMP production. The contractile response to (-)-norepinephrine (NE) was inhibited in the presence of IS or CLEN in control aortic rings. However, this modulatory effect was not seen in arteries from CLEN-treated rats. Preincubation of the arteries with either cholera toxin (an activator of the stimulatory guanine nucleotide binding protein, Gs) or forskolin reduced NE-induced vasoconstriction to the same extent in aortic rings from both control and CLEN-treated rats. The chronotropic response to NE in rat atria (beta-1-mediated) was not affected by the treatment. These results suggest that prolonged administration of CLEN to rats induced desensitization of beta-2 adrenoceptor-mediated vascular relaxation by alterations at the level of the beta-2 adrenergic receptor, but not in the mechanisms related to Gs, adenylate cyclase or in those distal to cAMP production.

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The importance of the interaction of alpha- and beta-adrenoceptors in maintaining vascular tone in rats was studied. This interaction after clenbuterol (CLEN) treatment indicates an important contribution of the circulating epinephrine (EPI) levels. In urethane-anesthetized rats, the beta 2-adrenoceptor antagonist ICI 118.551 was more effective in antagonizing isoproterenol-induced hypotension (mainly beta 2-mediated) than tachycardia (mainly beta 1-mediated). Intravenous (i.v.) administration of the alpha 2-adrenoceptor agonist clonidine (CLO) induced an initial pressor response followed by a more prolonged hypotension and bradycardia. The initial hypertensive effect was potentiated by previous acute administration of ICI 118.551 as well as by the nonselective beta-adrenoceptor antagonist propranolol, but not by metoprolol, a more selective beta 1-blocker. Fourteen days of administration of the beta 2-adrenoceptor agonist CLEN [0.3 mg/kg, subcutaneously (s.c.) twice daily], a treatment that induces desensitization of beta 2-mediated vasodilation, increased the pressor response induced by CLO, an effect that was not observed in pentobarbital-anesthetized rats. In any case, neither beta-blockers nor CLEN treatment affects the hypotension and bradycardia induced by CLO. Mean blood pressure (BP) of CLEN-treated rats was increased under urethane anesthesia but not under pentobarbital anesthesia. Catecholamine levels (principally EPI) were higher in urethane-anesthetized rats. These results provide further evidence of a functional interaction between alpha 2- and beta 2-adrenoceptor-mediated responses in rat vasculature and suggest that vasodilator beta 2-adrenoceptors might contribute to the determination of peripheral vascular tone when circulating EPI is substantially elevated.

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The effect of pretreatment with the beta-2-selective adrenoceptor agonist, (+/-)-clenbuterol (0.3 mg/kg, twice daily, 14 days) on prejunctional alpha-2- and beta-adrenoceptors was studied in rat atria. When atria from non-pretreated rats had been preincubated with (3H)-noradrenaline, (-)-isoprenaline (0.02 to 4.0 microM) did not affect tritium overflow evoked by stimulation of the cardioaccelerant nerves, but a higher concentration (40 microM) decreased it. Blockade of prejunctional inhibitory alpha-2-adrenoceptors by yohimbine (0.03, 0.3 and 0.8 microM) enhanced the overflow of tritium. In the presence of yohimbine, isoprenaline (1.2 microM) significantly increased stimulation-induced transmitter overflow, suggesting that in rat atria the facilitatory effect of isoprenaline mediated via prejunctional beta-adrenoceptors, is masked by the dominant influence of inhibitory alpha-2-adrenoceptors. (-)-Propranolol (0.1 microM) prevented the isoprenaline-induced increase in atrial rate and the isoprenaline-induced enhancement of transmitter release in the presence of yohimbine (0.3 microM), but did not modify by itself the stimulation-induced efflux of tritium, suggesting that neuronally released noradrenaline failed to activate facilitatory prejunctional beta-adrenoceptors. When atria from clenbuterol-pretreated rats had been preincubated with 3H-noradrenaline, the facilitatory effect of yohimbine 0.03 and 0.3 microM was markedly enhanced and, in this case, isoprenaline (1.2 and 12.0 microM) failed to cause its facilitatory effect in the presence of the alpha-2-adrenoceptor antagonist. Propranolol did not modify the facilitatory effect of yohimbine. No changes in the isoprenaline-induced increase in atrial rate were observed in clenbuterol-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

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Short-term treatment with clenbuterol [0.6 mg/kg-1 subcutaneously (s.c.) daily] produces a pressor effect in stressed rats after a period of immobilization (40 min). The stress applied markedly increases the plasma concentrations of norepinephrine (NE) and epinephrine. After bilateral adrenal demedullation, the increased levels of catecholamines and the hypertensive response obtained after clenbuterol treatment in the stressed animals were reduced to the values of the control rats. Clenbuterol treatment produced desensitization of the beta 2-adrenoceptor-mediated effect and thus reduced the vasodilator response induced by isoproterenol and increased the vasoconstriction produced by epinephrine but not that caused by NE. This desensitization may be responsible for the hypertensive response after clenbuterol treatment in stressed animals which is attenuated after adrenal demedullation. In conclusion, our results provide evidence that clenbuterol treatment induces pressor effect in normotensive animals under stress.

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