We sought to determine if different beta-adrenergic receptor (betaAR) subtypes, and their associated signalling machinery, are functionally localized to nuclear membranes. Employing enriched nuclear preparations, we assayed the specific presence of betaAR by measuring 125I-cyanopindolol (CYP) binding, Western blotting, confocal microscopy and functional assays. Western blots of rat heart nuclear fractions and confocal immunofluorescent analysis of adult rat and mouse ventricular cardiomyocytes displayed the presence of beta 1AR and beta 3AR but, surprisingly, not the beta 2AR on nuclear membranes. Nuclear localization of downstream signalling partners Gs, Gi and adenylyl cyclases II and V/VI was also demonstrated. The functional relevance of nuclear betaAR was shown by receptor-mediated stimulation of adenylyl cyclase activity by isoproterenol but not the beta 3AR-selective agonist CL 316243 in enriched nuclear preparations. We also examined the effect of subtype-selective ligands on the initiation of RNA synthesis in isolated nuclei. Both isoproterenol and another beta 3AR-selective agonist, BRL 37344, increased RNA synthesis which was inhibited by pertussis toxin (PTX). Neither a beta 1AR-selective agonist, xamoterol, nor a beta 2AR-selective agonist, procaterol, was able to stimulate transcription. However, both CGP 20712A and ICI 118,551 blocked isoproterenol-mediated effects to varying extents. PTX treatment also revealed that nuclear betaAR may be coupled to other signalling pathways in addition to Gi, as stimulation under these conditions reduced initiation of transcription below basal levels. These results highlight differential subcellular localization for betaAR subtypes and indicate that betaAR may have specific roles in regulating nuclear function in cardiomyocytes.

Endothelins are thought to act through two specific, plasmalemmal G protein-coupled receptor subtypes, ET_AR and ET_BR. However, in subfractionated cardiac membranes, ET_AR immunoreactivity was detected only in the plasma membrane whereas ET_BR immunoreactivity was detected predominantly in membranes of intracellular origin. Confocal microscopy demonstrated the presence of intracellular ET_AR and ET_BR in ventricular myocytes. ET_AR were primarily on plasma membrane (surface membranes and transverse-tubules) and to a lesser extent on the nucleus while ET_BR localized primarily to the nuclei. Western blot analysis of nuclei isolated from the heart indicated the presence of endothelin receptors: both ET_AR and ET_BR copurified with nucleoporin 62, whereas markers of endoplasmic reticulum and Golgi membranes were depleted. Radioligand binding studies revealed that isolated nuclei contain specific [¹²⁵I]ET-1 binding sites. Specific [¹²⁵I]ET-1 binding was reduced by 70–80% using the ET_AR-selective antagonist BQ610 and 20–30% using the ET_BR-specific antagonist BQ788. IRL-1620, an ET_BR-specific agonist, also reduced [¹²⁵I]ET-1 binding. Furthermore, ET-1 and IRL-1620 altered the incorporation of ³²P into nuclear proteins and caused a transient increase in nuclear Ca²⁺ concentration. Hence, cardiac nuclei possess both ET_AR and ET_BR subtypes, which are functional with respect to ligand binding and are coupled to signaling mechanisms within the nuclear membrane.

Tumour-promoting phorbol esters induce slow, sustained contractions of vascular smooth muscle, suggesting that protein kinase C (PKC) may play a role in the regulation of smooth muscle contractility. In some cases, e.g., ferret aortic smooth muscle, phorbol ester induced contractions occur without a change in [Ca²⁺]_i or myosin phosphorylation. Direct evidence for the involvement of PKC came from the use of single saponin-permeabilized ferret aortic cells. A constitutively active catalytic fragment of PKC induced a slow, sustained contraction similar to that triggered by phenylephrine. Both responses were abolished by a peptide inhibitor of PKC. Contractions of similar magnitude occurred even when the [Ca²⁺] was reduced to close to zero, implicating a Ca²⁺-independent isoenzyme of PKC. Of the two Ca²⁺-independent PKC isoenzymes, ε and ζ, identified in ferret aorta, PKCε is more likely to mediate the contractile response because (i) PKCε, but not PKCζ, is responsive to phorbol esters; (ii) upon stimulation with phenylephrine, PKCε translocates from the sarcoplasm to the sarcolemma, whereas PKCζ translocates from a perinuclear localization to the interior of the nucleus; and (iii) when added to permeabilized single cells of the ferret aorta at pCa 9, PKCε, but not PKCζ, induced a contractile response similar to that induced by phenylephrine. A possible substrate of PKCε is the smooth muscle specific, thin filament associated protein, calponin. Calponin is phosphorylated in intact smooth muscle strips in response to carbachol, endothelin-1, phorbol esters, or okadaic acid. Phosphorylation of calponin in vitro by PKC (a mixture of α, β, and γ isoenzymes) dramatically reduces its affinity for F-actin and alleviates its inhibition of the cross-bridge cycling rate. Calponin is phosphorylated in vitro by PKCε but is a very poor substrate of PKCζ. A signal transduction pathway is proposed to explain Ca²⁺-independent contraction of ferret aorta whereby extracellular signals trigger diacylglycerol production without a Ca²⁺ transient. The consequent activation of PKCε would result in calponin phosphorylation, its release from the thin filaments, and alleviation of inhibition of cross-bridge cycling. Slow, sustained contraction then results from a slow rate of cross-bridge cycling because of the basal level of myosin light chain phosphorylation (≈0.1 mol P_i/mol light chain). We also suggest that signal transduction through PKCε is a component of contractile responses triggered by agonists that activate phosphoinositide turnover; this may explain why smooth muscles often develop more force in response, e.g., to α₁-adrenergic agonists than to K⁺.Key words: smooth muscle, protein kinase C, calponin.

The effects of sex hormones on the heart are dependent on the presence and distribution of sex steroid hormone receptors (SSHR) in cardiac tissue. This study used subcellular fractionation, Western blot analysis and densitometry to characterize the subcellular distribution and abundance of estrogen receptor (ER) alpha, ERbeta and androgen receptor (AR) in atrial and ventricular tissue from male and female mice. The results showed that in both atrial and ventricular tissue ERalpha was primarily found in the sarcolemma, whereas ERbeta and AR were predominantly located in the nucleus and cytosol. Interestingly, ERalpha expression was greater in the ventricles compared to the atria, whereas ERbeta and AR expression were similar in both heart chambers. Furthermore, the distribution and abundance of SSHR in the atria and ventricles did not differ between sexes. This study also showed that a reduction in hormone levels (as a result of ovariectomy) resulted in a significant increase in the abundance of ERalpha in the ventricular sarcolemmal fraction. Overall, the results suggest ERalpha, ERbeta and AR distribution and expression are not sex dependent in the mouse heart. However, it appears that ERalpha expression is chamber specific and that, in certain cases, hormone levels can modulate the subcellular location of SSHRs.