The role of aging on contraction or relaxation through muscarinic or alpha-adrenergic receptors, respectively, was studied in isolated rat jejunum. Furthermore, the influence of extracellular calcium was analyzed, through functional and radioligand binding assays. The rank order of potency for selective muscarinic antagonists for M(1), M(2), and M(3) receptor subtypes, measured from affinity (pA(2)) values, was p-fluorohexahydrosiladifenidol (pFHHSiD) (M(3)) > pirenzepine (M(1)) > methoctramine (M(2)), indicating a predominance of M(3) subtype. This order was unchanged with age. Contractions by muscarinic agonist methacholine (MCh) were diminished in aged rats, resulting in lower apparent affinity (pD(2)) values, compared with adult controls. A larger decrease of MCh contractions occurred in aged rats after Ca(2+) withdrawal or after the calcium channel blocker isradipine. Changes were not detected for relaxation by adrenergic agonists. In conclusion, aging caused a decrease of MCh potency, which is probably related to the reduction of calcium sensitivity in jejunum.

Background and purpose: Androgens cause non-genomic relaxation in several smooth muscle preparations. However, such an effect has not been investigated in rat vas deferens yet. Our purpose was to study the effect of testosterone and derivatives in this tissue. Experimental approach: The influence of androgens was tested on contraction and translocation of intracellular Ca 2 þ induced by KCl in rat vas deferens in vitro. Key results: The testosterone derivative 5a-dihydrotestosterone produced a rapid and reversible concentration-dependent relaxation of KCl-induced contractions. Other androgens were also effective, showing the following rank order of potency: androsterone 45b-dihydrotestosterone 4androstenedione 45a-dihydrotestosterone 4testosterone. Calcium-induced contractions were also inhibited (about 45%) by 5a-dihydrotestosterone (30 mM). Moreover 5a-dihydrotestosterone blocked the increase of intracellular Ca 2 þ induced by KCl, measured by the fluorescent dye fura-2. Relaxation to 5a-dihydrotestosterone was resistant to the K þ channel antagonists glibenclamide, 4-aminopyridine and charybdotoxin. It was not affected by removal of epithelium or by L-NNA (300 mM), an inhibitor of nitric oxide biosynthesis, nor by selective inhibitors of soluble guanylate cyclase, ODQ or LY 83583, indicating that nitrergic or cGMP mediated mechanisms were not involved. The androgen-induced relaxation was also not blocked by the protein synthesis inhibitor cycloheximide (300 mM) or by the classical androgen receptor flutamide (up to 100 mM), corroborating that the effect is non-genomic. Conclusions and implications: Testosterone derivatives caused relaxation of the rat vas deferens, that did not involve epithelial tissue, K þ channels, or nitric oxide-dependent mechanisms, but was related to a partial blockade of Ca 2 þ influx.

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It has been almost 4 years since we revealed the explanation for the enigma of the so-called "calcium paradox". Our discovery of the involvement of Ca 2+ /cAMP signaling interaction in the regulation of neurotransmitter release, and neuroprotection, was clearly a serendipitous discovery. It has produced new avenues in the understanding of the cellular and molecular mechanisms involved in the pathogenesis of neurological and psychiatric disorders, such as Alzheimer´s disease. Interestingly, this discovery initiated decades ago when numerous clinical studies have reported that use of L-type Ca 2+ channel blockers (CCBs) by hypertensive patients decreased arterial pressure, but produced typical symptoms of sympathetic hyperactivity, such as tachycardia and increment of catecholamine plasma levels. Despite these adverse effects of CCBs have been initially attributed to adjust reflex of arterial pressure, during almost four decades this enigmatic phenomenon (the so-called "calcium paradox") remained unclear. In 2013, through an ingenious experiment, we discovered that this phenomenon was resulting of increment of transmitter release from sympathetic neurons, and adrenal chromaffin cells, stimulated by CCBs due to its interference on the Ca 2+ /cAMP signaling interaction. In this way, our discovery of the role of Ca 2+ /cAMP intracellular signaling interaction in the neurotransmitter release, and neuronal death triggered by cytosolic Ca 2+ overload, opened novel adventures for the development of new pharmacological strategies more effective for the treatment of neurological and psychiatric disorders resulting of neurotransmitter release deficit, and neuronal death. These novel concepts have been extensively documented in several cited international papers of our own authorship (Bergantin and Caricati-Neto), and in an international book.

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