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Objective: This study was undertaken to investigate in human tissue samples the mechanisms underlying spontaneous and prostaglandin F(2)(alpha)-induced contractions during the final trimester of pregnancy and labor.Study Design: Membrane potential and cytoplasmic calcium were recorded simultaneously with contraction in uterine strips obtained from the lower segment during cesarean delivery.Results: Between week 28 of gestation and term there was a progressive increase in the frequency of spontaneous contractions and a decrease in the negative potential of the membrane. The response to prostaglandin F(2alpha) was biphasic. The initial excitatory component remained stable toward term. A later inhibitory component, which was underpinned by increased activity of the sodium-potassium adenosine triphosphatase pump, decreased at the time of labor.Conclusions: There is a gradual increase in excitability in uterine muscle throughout the third trimester of human pregnancy. The initial component of the prostaglandin response is a large contraction that is kept brief by a subsequent inhibitory component of the response, which ensures that full relaxation occurs between contractions. [ABSTRACT FROM AUTHOR]

Polymyxin antibiotics are often used as a last-line defense to treat life-threatening Gram-negative pathogens. However, polymyxin-induced kidney toxicity is a dose-limiting factor of paramount importance and can lead to suboptimal treatment. To elucidate the mechanism and develop effective strategies to overcome polymyxin toxicity, we employed a whole-genome CRISPR screen in human kidney tubular HK-2 cells and identified 86 significant genes that upon knock-out rescued polymyxin-induced toxicity. Specifically, we discovered that knockout of the inwardly rectifying potassium channels Kir4.2 and Kir5.1 (encoded by KCNJ15 and KCNJ16, respectively) rescued polymyxin-induced toxicity in HK-2 cells. Furthermore, we found that polymyxins induced cell depolarization via Kir4.2 and Kir5.1 and a significant cellular uptake of polymyxins was evident. All-atom molecular dynamics simulations revealed that polymyxin B 1 spontaneously bound to Kir4.2, thereby increasing opening of the channel, resulting in a potassium influx, and changes of the membrane potential. Consistent with these findings, small molecule inhibitors (BaCl 2 and VU0134992) of Kir potassium channels reduced polymyxin-induced toxicity in cell culture and mouse explant kidney tissue. Our findings provide critical mechanistic information that will help attenuate polymyxin-induced nephrotoxicity in patients and facilitate the design of novel, safer polymyxins., (© 2022. The Author(s).)

Nkx2-5 is one of the master regulators of cardiac development, homeostasis and disease. This transcription factor has been previously associated with a suite of cardiac congenital malformations and impairment of electrical activity. When disease causative mutations in transcription factors are considered, NKX2-5 gene dysfunction is the most common abnormality found in patients. Here we describe a novel mouse model and subsequent implications of Nkx2-5 loss for aspects of myocardial electrical activity. In this work we have engineered a new Nkx2-5 conditional knockout mouse in which flox sites flank the entire Nkx2-5 locus, and validated this line for the study of heart development, differentiation and disease using a full deletion strategy. While our homozygous knockout mice show typical embryonic malformations previously described for the lack of the Nkx2-5 gene, hearts of heterozygous adult mice show moderate morphological and functional abnormalities that are sufficient to sustain blood supply demands under homeostatic conditions. This study further reveals intriguing aspects of Nkx2-5 function in the control of cardiac electrical activity. Using a combination of mouse genetics, biochemistry, molecular and cell biology, we demonstrate that Nkx2-5 regulates the gene encoding Kcnh2 channel and others, shedding light on potential mechanisms generating electrical abnormalities observed in patients bearing NKX2-5 dysfunction and opening opportunities to the study of novel therapeutic targets for anti-arrhythmogenic therapies., (Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

The hypothesis tested in this study is that diabetes has a different impact on an artery in which endothelium-dependent responses derive from both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) compared with responses in which NO predominates and EDHF is absent. The streptozotocin-treated rat model of diabetes was used, and the arteries were mounted on a wire myograph. In mesenteric arteries depolarized and constricted with phenylephrine, acetylcholine evoked hyperpolarization (31 +/- 2 mV) and complete relaxation; these responses were attributed to EDHF and NO. In femoral arteries, acetylcholine evoked a small, NO-mediated hyperpolarization (5 +/- 1 mV) and incomplete relaxation. Bradykinin evoked NO-dependent responses in mesenteric arteries. Whereas diabetes significantly impaired the EDHF-dependent hyperpolarization and relaxation in mesenteric arteries, NO-dependent responses in femoral and mesenteric arteries were preserved. 1-Ethyl-2-benzimidazolinone evoked hyperpolarization and relaxation in mesenteric arteries, and this was impaired in diabetes. In conclusion, NO-dependent responses are preserved in diabetes, whereas endothelial responses-dependent upon EDHF appear to be impaired. The putative channels responsible for mediating the EDHF response may be altered in diabetes.

1. The mechanisms involved in contraction in guinea-pig myometrium were compared at mid- and late pregnancy. Tension was recorded simultaneously with either membrane potential or cytoplasmic calcium ([Ca2+]i) in strips exposed briefly to prostaglandin F2alpha (PGF). 2. PGF-induced increases in tension were underpinned by action potentials followed by sustained depolarization and biphasic increases in [Ca2+]i at mid- (peak, 879 +/- 199 nM; sustained, 298 +/- 35 nM, n = 11) and late pregnancy (peak, 989 +/- 302 nM; sustained 178 +/- 33 nM, n = 8). 3. At mid- and late pregnancy, nifedipine (10-6 M) reduced (a) the PGF-induced increase in tension to 84 and 35 %, (b) the level attained during the depolarization by 2 and 12 mV and (c) the peak rise in [Ca2+]i to 42 and 17 %. The sustained rises in [Ca2+]i were resistant to nifedipine. 4. In Ca2+-free solution (containing 1 mM EGTA), PGF elicited an increase in tension that was 26 % of that in 2.5 mM Ca2+ and an increase in [Ca2+]i (24 % of the sustained level) at mid-pregnancy but no increase in tension or [Ca2+]i at term. 5. At both stages of pregnancy, PGF decreased the level of [Ca2+]i required to elicit increases in tension comparable to those evoked by high K+o. The slope of the tension-[Ca2+]i curves were steeper in mid- than in late pregnancy. 6. In conclusion, at mid-pregnancy, the contractile response of the guinea-pig myometrium to PGF involves Ca2+ influx through L-type voltage-operated Ca2+ channels (VOCCs) and by receptor-operated mechanisms, release of Ca2+ from intracellular stores, and an increase in the sensitivity of the contractile apparatus to Ca2+. At term the situation is different: a modest increase in the sensitivity of the contractile apparatus to Ca2+ persists and there is a major reliance on Ca2+ influx through VOCCs.

1. The effects of prostaglandins E2 (PGE) and F2alpha (PGF) on membrane potential and isometric tension and cytoplasmic free calcium concentration ([Ca2+]i) and tension were studied in strips of uterine smooth muscle obtained from women undergoing Caesarean delivery at term and during established labour. 2. Prostaglandins (PGs) evoked a biphasic response. The excitatory component consisted of depolarization of the membrane, which initiated spike action potentials, an increase in [Ca2+]i and tension development. The membrane remained depolarized at -19 +/- 1 mV for about 2 min, then repolarized abruptly, [Ca2+]i promptly returned to basal levels, and tension development ceased. 3. This component of the response to PGE or PGF was followed by a slow hyperpolarization which reached -85 +/- 2 mV (n = 22) at term and -70 +/- 2 mV (n = 9) during labour, and during which spontaneous action potentials and tension development did not occur. 4. Nifedipine (10-6 M) abolished spontaneous activity, abolished PG-induced action potentials and reduced the increase in [Ca2+]i (9 +/- 3 %, n = 6), the depolarization (10 +/- 1 mV, n = 14), the tension (2 +/- 1 %, n = 14) and the hyperpolarization (9 +/- 1 mV, n = 14, at term). 5. A variety of K+ channel blockers were without effect on the peak amplitude of the PG-induced hyperpolarization but the latter did not occur in the presence of ouabain (10-6 M) or in K+-free or low-Na+ solutions, suggesting an involvement of the Na+-K+-ATPase pump. 6. In conclusion, a substantial dependence on Ca2+ influx through voltage-operated Ca2+ channels accounts for the importance of membrane potential in regulating contractions in human uterine smooth muscle. The classical excitatory effect of PGE and PGF is followed by hyperpolarization involving the Na+-K+-ATPase pump. The hyperpolarization restricts the response to a single contraction and decreases the frequency of subsequent contractions. The amplitude of the hyperpolarization decreases during labour, allowing contraction frequency to increase. Its persistence at this time ensures complete relaxation between each single robust contraction, preventing spasm of the uterus that would restrict blood flow to the fetus during delivery.

Membrane potentials were recorded with conventional intracellular microelectrodes from chromaffin cells in isolated, bisected adrenal glands from guinea-pigs. The local pressure ejection of muscarinic agonists, acetylcholine (in the presence of hexamethonium) or bethanecol, caused a transient depolarization that was relatively slow (1-2 s) in onset compared with the depolarization associated with the activation of nicotinic receptors. Muscarinic receptor-induced depolarization was associated with an increase in input resistance and the firing of action potentials. Repetitive stimulation of splanchnic nerve fibers within the gland, in the presence of hexamethonium, caused a maintained depolarization that was slow in both onset and decay and in many cells caused the repetitive firing of action potentials. It is suggested that, in this species, the exocytosis of catecholamines caused by the activation of muscarinic receptors, described by others, may be due to the initiation of tetrodotoxin-sensitive action potentials and consequent opening of voltage sensitive Ca2+ channels.

The effects of stimulating the intramural nerves on the membrane potential and tension in the uterine artery of virgin guinea pigs were compared with the responses during pregnancy. In all tissues the amplitude of the excitatory junction potential (EJP) increased as the stimulus voltage was increased. The rate of increase in EJP amplitude in tissues from virgin animals greatly exceeded that recorded in late pregnant tissues. EJPs were abolished by tetrodotoxin but were resistant to blockade by alpha-adrenoceptor antagonists. Stimulation of the nerves also evoked a slow depolarization and contraction which were abolished by both tetrodotoxin and alpha-adrenoceptor antagonists. The amplitudes of the depolarizations and contractions were not correlated. The role of EJPs and alpha-adrenoceptor activation in the control of vascular function is discussed. Fluorescence histochemistry revealed a decrease in the density of the catecholamine innervation that was correlated with a decrease in catecholamine content as pregnancy progressed. In addition, there appeared to be a difference in the arrangement of the fluorescent varicosities, with a shift from varicosities that were close to the outer layer of smooth muscle in virgin tissues to those that were more distantly dispersed in the adventitia during late pregnancy. The changes would be expected to reduce the effectiveness of vasoconstrictor drive to the uterine artery as pregnancy progresses.

Action potentials were recorded during impalements of some but not all smooth muscle cells of mouse vas deferens in response to both nerve stimulation and intracellular current injection. They were resistant to blockade by nifedipine (0.1-1.0 microM) but were blocked by tetrodotoxin (TTX, 0.2-1.0 microM) when this was added in the presence of nifedipine. It is suggested that voltage-dependent sodium (Na+) channels are present in mouse vas deferens that function to amplify calcium (Ca2+) influx through voltage-dependent Ca2+ channels.

1. The partial muscarinic agonist, pilocarpine, evoked concentration-dependent relaxation with an EC50 of 2.4 x 10(-3) M in isolated segments of rat tail artery that were constricted with phenylephrine (10(-8) to 2 x 10(-7) M). Acetylcholine also evoked concentration-dependent relaxation but was more potent than pilocarpine (EC50, 6.5 x 10(-7) M). 2. The concentration-relaxation curves for pilocarpine were not affected by the muscarinic antagonists, atropine (10(-9) M) or pirenzepine (5 x 10(-7) M), while the concentration-relaxation curves for acetylcholine-evoked relaxation of the same tissues were shifted some 10 fold to the right by these concentrations of atropine and pirenzepine. 3. Acetylcholine failed to evoke relaxation following removal of the endothelium. The smooth muscle of the rat tail artery was some 10 fold more sensitive to the relaxing action of pilocarpine following denudation of the endothelium. 4. The effects of pilocarpine and acetylcholine on membrane potential were studied in tissues that were depolarized to -39 +/- 1 mV with phenylephrine (5 x 10(-8) to 2 x 10(-7) M). In intact tissues, pilocarpine caused hyperpolarization, an effect that persisted in the presence of muscarinic antagonists. Acetylcholine also evoked hyperpolarization. 5. Following removal of the endothelium, pilocarpine (10(-5) to 10(-3) M) evoked hyperpolarization in 6 of 15 preparations and a decrease in the frequency of action potentials in the remainder. Both of these responses were associated with relaxation. 6. The effects of pilocarpine were compared with other agents that evoke endothelium-independent relaxation. The concentration-relaxation curves in response to pilocarpine and nitroprusside were shifted to the right by ferricyanide (10-5 M) and methylene blue (10-5 M). Glibenclamide (10-6 M) was without effect on the hyperpolarization and relaxation evoked by pilocarpine (10' to 10- M).7. Thus, pilocarpine evokes relaxation of rat tail artery independently of the cholinergic system and it is suggested that this is achieved by decreasing the frequency of action potentials in the smooth muscle.