Luis M. Montaño

Androgens induce relaxation of contractile activity in pregnant human myometrium at term: a nongenomic action on L-type calcium channels

Abstract It has long been accepted that progesterone regulates uterine contractile activity. However, little is known about the role of androgens in female physiology, and their importance and biological function on myometrial contractility so far have received limited attention. In this work, we examined the direct effect of androgens on the contractile activity of the isolated human myometrium. Myometrial biopsies were obtained, with consent, from pregnant women undergoing elective cesarean section at term. Each androgen tested (dehydroepiandrosterone, testosterone, 5α- and 5β-dihydrotestosterone, androsterone, or androstanediol) caused a concentration-dependent inhibition of spontaneous contractile activity; a relaxing effect of these androgens was also observed on the contractions induced by high potassium (KCl) solution. Interestingly, nonpregnant myometrium was also sensitive to androgen-induced relaxation. 5β-Dihydrotestosterone (5β-DHT) was dramatically more potent than the other androgens in inducing myometrial relaxation in all preparations. Relaxation response to androgens had very rapid time courses and was affected by neither the specific antiandrogen (flutamide) nor inhibitors of protein synthesis (cycloheximide) and transcription (actinomycin D), implying that androgens act through a nongenomic mechanism. Importantly, 5β-DHT significantly reduced the increase in intracellular calcium concentration associated with exposure to KCl in human myometrial smooth-muscle cells loaded with Fura-2-AM. The blockade of l-type calcium channels seems to be involved in the nongenomic relaxing action of androgens. These observations demonstrate that androgens may play a crucial role in maintaining pregnancy.

Cross-inhibitory interactions between GABAA and P2X channels in myenteric neurones

Inhibitory interactions between GABAA[induced by γ‐aminobutyric acid (GABA)] and P2X [activated by adenosine 5′‐triphosphate (ATP)] receptors of myenteric neurones from the guinea pig small intestine were characterized using whole‐cell recordings. Currents induced by GABA (IGABA) or ATP (IATP) were inhibited by picrotoxin or pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid, respectively. Currents induced by GABAu2003+u2003ATP (IGABA+ATP) were only as large as the current induced by the most effective transmitter, revealing current occlusion. This occlusion requires maximal activation of at least one of these receptors. Sequential applications of neurotransmitters, and kinetic and pharmacological properties of IGABA+ATP indicate that they are carried through both GABAA and P2X channels. ATP did not affect IGABA in neurones: (i) in which P2X channels were not present; (ii) after inhibiting P2X channels with Ca2+ (iii) in the presence of pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid, a P2X receptor antagonist; (iv) after P2X receptor desensitization or (v) at IATP reversal potential. Similarly, GABA did not affect P2X‐mediated currents in neurones: (i) in which GABAA channels were not present; (ii) in the presence of picrotoxin, a GABAA channel blocker; (iii) after GABAA receptor desensitization or (iv) at the IGABA reversal potential. Current occlusion occurred as fast as current activation and it was still present in the absence of Ca2+, at 11u2003°C, after adding to the pipette solution a cocktail of protein kinase inhibitors (staurosporineu2003+u2003genisteinu2003+u2003K‐252a), after substituting the GTP in the pipette with GDP‐β‐S and after treating the cells with N‐ethylmaleimide. Taken together, all of these results are consistent with a model of cross‐inhibition between GABAA and P2X.

In airways ATP refills sarcoplasmic reticulum via P2X smooth muscle receptors and induces contraction through P2Y epithelial receptors

In airway smooth muscle (ASM), ATP induces a contraction associated with the increase of [Ca2+]i. Cytosolic Ca2+ is extruded to the extracellular space by the Na+/Ca2+ exchanger (NCX) in its normal mode. Some agonists activate the reverse mode of the NCX (NCXREV), inducing Ca2+ entry. We investigated whether ATP, via P2X receptors, activates the NCXREV and whether the increment in [Ca2+]i is used for contraction or for the sarcoplasmic reticulum (SR) refilling in guinea pig ASM. ATP contracted the ASM and this effect was blocked by indomethacin. Suramin and RB2 diminished the contraction induced by ATP; PPADS did not modify this response. In myocytes, ATP produces an increase in [Ca2+]i not modified by indomethacin. In tracheal strips, using simultaneous measurements, ATP induced a biphasic change in [Ca2+]i, (a Ca2+ peak followed by a plateau) accompanied by a contraction. Indomethacin or epithelium removal abolished this contraction, but not the Ca2+ peak, whereas the plateau was decreased by indomethacin. In myocytes, the ATP-induced [Ca2+]i increment was inhibited by suramin (~96%), PPADS (~40%), and RB2 (~57%). ATP augmented the NCXREV and this effect was abolished by SKF 96365 and TNP-ATP (P2X1 and P2X3 receptors antagonist). P2X1 and P2X3 receptors were corroborated by immunoblotting of ASM. NCXREV activation and ATP in the presence of RB2 favor the SR Ca2+ refilling. In tracheal rings, successive ATP stimulations were reduced with KB-R7943. Therefore, ATP: (1) indirectly promotes muscle contraction via epithelial P2Y receptors and prostaglandins release; (2) increases the [Ca2+]i through a prostaglandin-independent manner by activating P2X and P2Y receptors in smooth muscle; and (3) activates P2X1 and P2X3 receptors and the NCXREV which refills the SR.

Sarcoplasmic reticulum Ca2+ refilling is determined by L-type Ca2+ and store operated Ca2+ channels in guinea pig airway smooth muscle

Sarcoplasmic reticulum Ca(2+) refilling (SRREF) is crucial to sustain the agonists induced airway smooth muscle contraction. Nevertheless, its mechanisms have not been clearly described yet, although L-type voltage dependent, store operated, receptor operated channels and the Na(+)/Ca(2+) exchanger in its reverse mode (NCXREV) have been proposed as Ca(2+) handling proteins participating in this process. We found that in guinea pig and bovine tracheal smooth muscle, SRREF induced by caffeine was completely abolished by thapsigargin, even in the presence of Bay K8644, an activator of the L-type Ca(2+) channel. Activation of NCXREV in guinea pig tracheal myocytes increased SRREF in ~70%, while opening of the L-type Ca(2+) channels with Bay K8644 and favoring the capacitative Ca(2+) entry with 2-APB (32 μM) also augmented the SRREF by ~170% and ~71%, respectively. Methoxyverapamil (D-600, an L-type Ca(2+) channel blocker), 2-APB (100 µM, antagonist of the capacitative Ca(2+) entry) and PPADS (NCXREV blocker) diminished the SRREF by ~63%, ~72% and ~31%, respectively. The simultaneous addition of D-600 and 2-APB annulled SRREF. These last results were also seen when carbachol was used instead of caffeine. In tracheal rings, 2-APB and nifedipine abolished the carbachol-induced contraction. We concluded that the sarcoplasmic reticulum Ca(2+) pump is the only mechanism involved in the SRREF and that L-type Ca(2+) voltage dependent and store operated Ca(2+) channels are the principal membranal Ca(2+) handling proteins that provide extracellular Ca(2+) for SRREF and carbachol-induced contraction in the guinea pig airway smooth muscle; NCXREV seems to play a minor role.

Androgens are bronchoactive drugs that act by relaxing airway smooth muscle and preventing bronchospasm

Changes in the androgen levels in asthmatic men may be associated with the severity of asthma. Androgens induce a nongenomic relaxation in airway smooth muscle, but the underlying mechanisms remain unclear. The aim of this study was to investigate the potential bronchorelaxing action of testosterone (TES) and its metabolites (5α- and 5β-dihydrotestosterone (DHT). A preventive effect on ovalbumin (OVA)-induced bronchospasm was observed in sensitized guinea pigs for each androgen. Androgens were studied in response to bronchoconstrictors: carbachol (CCh) and KCl in isolated trachea rings with and without epithelium from non-sensitized and sensitized animals as well as on OVA-induced contraction. Androgens concentration-dependently abolished the contraction in response to CCh, KCl, and OVA. There were significant differences in the sensitivity to the relaxation induced by each androgen. 5β-DHT was more potent for relaxing KCl-induced contraction, while TES and 5α-DHT were more potent for CCh- and OVA-induced contraction. No differences were found in preparations with and without epithelium or in the presence of a nitric oxide (NO) synthase inhibitor or an inhibitor of K(+) channels. These data indicate the absence of involvement of the epithelium-, NO- and K(+) channels-dependent pathway in androgen-induced relaxation. However, in dissociated tracheal myocytes loaded with the calcium-binding fluorescent dye Fura -2, physiological concentrations of androgens decreased the KCl-induced [Ca(2+)]i increment. 5β-DHT was the most potent at decreasing KCl-induced [Ca(2+)]i increment and preventing bronchospasm. We suggest that androgen-induced brochorelaxation was mediated via decreased Ca(2+) influx through L-type Ca(2+)channels but additional Ca(2+) entry blockade may be involved. Molecular changes in androgen structure may determine its preferential site of action.

Role of 5‐HT2A, 5‐HT4 and 5‐HT7 receptors in the antigen‐induced airway hyperresponsiveness in guinea‐pigs

Background A possible role of 5‐hydroxytryptamine (5‐HT) in the origin of antigen‐induced airway hyperresponsiveness (AI‐AHR) has been scarcely investigated.

Role of sarcoplasmic reticulum Ca2+ content in Ca2+entry of bovine airway smooth muscle cells

Depletion of intracellular Ca2+ stores induces the opening of an unknown Ca2+ entry pathway to the cell. We measured the intracellular free-Ca2+ concentration ([Ca2+]i) at different sarcoplasmic reticulum (SR) Ca2+ content in fura-2-loaded smooth muscle cells isolated from bovine tracheas. The absence of Ca2+ in the extracellular medium generated a time-dependent decrement in [Ca2+]i which was proportional to the reduction in the SR-Ca2+ content. This SR-Ca2+ level was indirectly determined by measuring the amount of Ca2+ released by caffeine. Ca2+ restoration at different times after Ca2+-free incubation (2, 4, 6 and 10xa0min) induced an increment of [Ca2+]i. This increase in [Ca2+]i was considered as Ca2+ entry to the cell. The rate of this entry was slow (~0.3xa0nM/s) when SR-Ca2+ content was higher than 50% (2 and 4xa0min in Ca2+-free medium), and significantly (p<0.01) accelerated (>1.0xa0nM/s) when SR-Ca2+ content was lower than 50% (6 and 10xa0min in Ca2+-free medium). Thapsigargin significantly induced a higher rate of this Ca2+ entry (p<0.01). Variations in Ca2+ influx after SR-Ca2+ depletion were estimated more directly by a Mn2+ quench approach. Ca2+ restoration to the medium 4xa0min after Ca2+ removal did not modify the Mn2+ influx. However, when Ca2+ was added after 10xa0min in Ca2+-free medium, an increment of Mn2+ influx was observed, corroborating an increase in Ca2+ entry. The fast Ca2+ influx was Ni2+ sensitive but was not affected by other known capacitative Ca2+ entry blockers such as La3+, Mg2+, SKF 96365 and 2-APB. It was also not affected by the blockage of L-type Ca2+ channels with methoxyverapamil or by the sustained K+-induced depolarisation. The slow Ca2+ influx was only sensitive to SKF 96365. In conclusion, our results indicate that in bovine airway smooth muscle cells Ca2+ influx after SR-Ca2+ depletion has two rates: A) The slow Ca2+ influx, which occurred in cells with more than 50% of their SR-Ca2+ content, is sensitive to SKF 96365 and appears to be a non-capacitative Ca2+ entry; and B) The fast Ca2+ influx, observed in cells with less than 50% of their SR-Ca2+ content, is probably a capacitative Ca2+ entry and was only Ni2+-sensitive.

Sarcoplasmic Reticulum Ca2+ Depletion by Caffeine and Changes of [Ca2+]i During Refilling in Bovine Airway Smooth Muscle Cells

BACKGROUNDnIn airway smooth muscle (ASM), Ca2+ influx in response to the Ca2+ depletion of the sarcoplasmic reticulum (SR) seems to play a role in the regulation of intracellular free Ca2+ concentrations ([Ca2+](i)). This study evaluates some possible Ca2+ entry pathways activated during SR-Ca2+ depletion induced by 10 mM caffeine.nnnMETHODSnEnzymatically dispersed bovine ASM cells were loaded with Fura-2/AM to permit measurement of [Ca2+](i) changes in single cells.nnnRESULTSnCaffeine (10 mM) induced a transient increase in [[Ca2+](i) that depleted SR-Ca(2)+ content. After caffeine washout, a decrease in basal [Ca2+](i) (undershoot) was invariably observed, followed by a slow recovery. This phenomenon was inhibited by cyclopiazonic acid (5 microM). External Ca(2)+ removal in depolarized and nondepolarized cells induced a decrease in basal [Ca2+](i) that continued until depletion of the SR-Ca2+ content. The decrease in [Ca2+](i) induced by Ca2+-free physiological saline solution (PSS) was accelerated in caffeine-stimulated cells. Recovery from undershoot was not observed in Ca2+-free PSS. Depolarization with KCl and addition of D600 (30 microM) did not modify recovery. Similar results were obtained when the Na(+)/Ca2+ exchanger was blocked by substituting NaCl with KCl in normal PSS (Na(+)-free PSS) or by adding benzamil amiloride (25 microM).nnnCONCLUSIONSnSR-Ca2+ content plays an important role in the Ca2+ leak induced by Ca2+-free medium, and does not depend on membrane potential. Additionally, recovery from undershoot after caffeine depends on extracellular Ca2+, and neither voltage-dependent Ca2+ channels nor the Na(+)/Ca2+ exchanger are involved.

Involvement of different Ca2+ pools during the canine bronchial sustained contraction in Ca2+-free medium: lack of effect of PKC inhibition

We evaluated the role of protein kinase C (PKC) in the sustained bronchial contraction (SBC) induced by carbachol (Cch) or histamine in a Ca2+-free medium and the possibility that each agonist uses a different Ca2+ store for this response. We studied third-order bronchi and airway smooth muscle (ASM) from first-order bronchi dissected free of cartilage and epithelium. Bronchial and ASM responsiveness to Cch or histamine were evaluated in Krebs solution (2.5 mM Ca2+) and in Ca2+-free medium. Cch and histamine induced an SBC in bronchial tissues in Ca2+-free medium. In ASM each agonist produced a transient contraction, but the response to histamine was much smaller. Cch induced a concentration-dependent accumulation of inositol phosphates (IPs) in both bronchi and ASM; however, histamine did not induce significant accumulation of IPs. Repeated exposure to histamine in bronchial rings abolished contractile responses in Ca2+-free media, but Cch added afterwards still produced a sustained contraction. This response was blocked when bronchial tissues were preincubated with 10 µM cyclopiazonic acid (CPA). Brief incubation of these preparations with a high EGTA concentration (1 mM) abolished the histamine-induced SBC. The SBC induced by Cch or histamine in Ca2+-free medium was not affected by the preincubation of the tissues with calphostin C, chelerythrine or staurosporine. We concluded that Cch mobilizes Ca2+ from two different sources during the SBC in Ca2+-free medium: from a CPA-sensitive one from sarcoplasmic reticulum (SR) and from a putative extracellular membrane Ca2+ pool sensitive to 1 mM EGTA, and neither process involved PKC activation. Histamine appeared to utilize the extracellular membrane pool only.

Regional differences in the vasorelaxing effects of testosterone and its 5-reduced metabolites in the canine vasculature

Although the vasorelaxing effects of testosterone (T) and various androgen metabolites have been observed in a variety of blood vessels and species, previous studies have not systematically compared the vasorelaxing effects of androgen metabolites in different vascular beds within the same species. Therefore, we studied the vasorelaxing effects of T and its 5-reduced metabolites (5α- and 5β-DHT) on KCl-induced contractions of the canine left coronary artery, femoral artery and saphenous vein, using standard isometric recordings. KCl contractions were inhibited by each androgen in a concentration-dependent manner from 1.8 to 310μM. Vascular sensitivity and efficacy were expressed as inhibitory concentration 50 (IC₅₀) and maximal relaxation (R(max)), respectively. The coronary artery was significantly more sensitive to androgen-induced vasorelaxation than the saphenous vein or femoral artery. These vasorelaxing responses were unaffected by an antiandrogen (Flutamide) or the sulfhydryl reagent, N-ethylmaleimide, suggesting a nongenomic mechanism independent of signaling mediated by the androgen receptor or G proteins. Concentration-response curves were unchanged in endothelium-denuded preparations; thus, the endothelium appears to have no role in androgen-induced vasorelaxation. 5β-DHT was the most potent androgen in both coronary and femoral artery, but all three androgens were equipotent in the saphenous vein. It is concluded that: 1) significant regional differences exist in vasorelaxing effects of androgen metabolites in the canine vasculature; 2) structural differences in these androgens determine their vasorelaxing efficacy; and 3) regional differences in androgen-induced vasorelaxation may account for some of the conflicting findings reported on the vasorelaxing effects of the androgens.