Effects of pH manipulation, CatSper stimulation and Ca2+-store mobilization on [Ca2+]i and behaviour of human sperm.

STUDY QUESTION: How do the alkaline pH, progesterone and 4-aminopyridine interact in their effects on human sperm? SUMMARY ANSWER: Behaviour of human sperm (proportion of hyperactivated cells and motility kinematics) were related directly to [Ca2+]i irrespective of pH or the agonist applied. WHAT IS KNOWN ALREADY?: CatSper channels of human sperm, which are central to generation of sperm [Ca2+]i signals and induction of hyperactivated motility, are activated by intracellular alkalinization and progesterone. Progesterone (P4) is much less effective than 4-aminopyridine (4-AP) (which mobilizes stored Ca2+ but also raises pHi) as an inducer of hyperactivation. STUDY DESIGN, SIZE, DURATION: This was a laboratory study, spanning ~18 months that used 15 sperm donors and involved more than 100 separate experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors and patients were recruited in accordance with local ethics approval (ERN_12-0570R). [Ca2+]i responses of suspended cell populations were examined by fluorimetric recording and motility parameters assessed by computer-assisted sperm analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Increasing pHo from 7.4 to 8.5 raised pHi (from 6.9 to 7.2) and significantly increased both [Ca2+]i and the proportion of hyperactivated cells. Stimulation of cells with P4 (1 nM-20 µM) induced a biphasic (transient and plateau) increase in [Ca2+]i. The [Ca2+]i increase was of similar amplitude and dose-dependency at pHo = 7.4 and pHo = 8.5. 4-aminopyridine (0.2-5 mM) induced a biphasic [Ca2+]i increase that was dose-dependent across the entire range tested and was strongly enhanced at pH 8.5. Motility was assessed 300 s post-stimulation, during the plateau phase of the progesterone and 4-AP-induced [Ca2+]i responses. Progesterone had only a small effect on hyperactivated motility even at the highest dose used (20 µM; < 5% increase in the proportion of cells classified as hyperactivated) which was insensitive to pHo. 4-Aminopyridine potently stimulated hyperactivated motility, this effect being dose-dependent and greatly enhanced at pHo = 8.5. The relationship between [Ca2+]i (fluorescence of fluo4) and proportion of hyperactivated cells, irrespective of pHo, agonist or dose, was fitted by a single curve (second order polynomial; R2 = 0.96). Similar analysis of curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) showed a linear relationship to [Ca2+]i (R2 > 0.9). LIMITATIONS, REASONS FOR CAUTION: This was an in-vitro study and caution must be taken when extrapolating these results to in vivo regulation of sperm. Though controls indicate that saturation of fluo4 did not affect the findings, at the highest doses of progesterone the true amplitude of the [Ca2+]i transient may not have been reported by the dye. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that (i) activation of human sperm CatSper by progesterone (and presumably other ligands that act similarly) and consequent acquisition of hyperactivated motility is not significantly enhanced by intracellular alkalinization; (ii) VCL, ALH and hyperactivation are directly related to [Ca2+]i, irrespective of the mechanism by which Ca2+ is mobilized, and the ability of stimuli to induce prolonged [Ca2+]i elevation (as occurs upon mobilization of stored Ca2+) determines the observed effect on cell behaviour. STUDY FUNDING/COMPETING INTEREST(S): CA was supported by the Nigerian government (Tertiary Education Trust (TET) Fund). The authors have no conflicts of interest.

Effects of age on expression of BKca channel in vascular smooth muscle cells from mesenteric arteries of spontaneously hypertensive rats

Abnormally functioning K+ channels during aging might contribute to increased arterial tone of vascular smooth muscle cells. However, changes in large conductance calcium-activated potassium (BKca) channel current density and channel protein composition during aging are not fully understood and are currently investigated here. Spontaneously hypertensive rats (SHR) and Wistar–Kyoto (WKY) rats were grouped by age into very young (4–6 weeks), young (16–18 weeks), and adult (40–42 weeks) rats. BKca current was measured using a whole cell patch clamp in vascular smooth muscle cells from rat mesenteric arteries. BKca channel and subunits were measured using confocal imaging and real-time reverse transcriptase polymerase chain reaction techniques. Systolic and diastolic blood pressure increased significantly with age in both SHR and WKY rats. The expression of BKca á subunits increased in plasma membrane and cytoplasm in both SHR and WKY rats during aging, but had a larger increase in the cytoplasm in SHR than in WKY rats. BKca current density increased with aging in SHR but not in WKY rats. The relative abundance of BKca áand â1 mRNA transcripts changed minimally or not at all with aging in both SHR and WKY rats. In conclusion, plasma membrane BKca protein increased with aging in both SHR and WKY rats. Increases in cytoplasmic BKca protein and in BKca current were greater in SHR than WKY rats (AU)

Effect of renal impairment on the pharmacokinetics of PD 0200390, a novel ligand for the voltage-gated calcium channel alpha-2-delta subunit.

AIMS: To investigate the pharmacokinetics and safety of PD 0200390 in healthy subjects and subjects with renal impairment (RI) and to examine the relationship between oral and renal PD 0200390 clearance and estimated creatinine clearance (CLcr). METHODS: In this open-label study, 26 subjects were categorized into four groups based on renal function: no RI (CLcr >80 ml min(-1); n= 6); mild RI (CLcr 51 to < or =80 ml min(-1); n= 6); moderate RI (CLcr >30 to 50 ml min(-1); n= 6); and severe RI (CLcr < or =30 ml min(-1); n= 8). Subjects received a single, oral dose of PD 0200390 25 mg. Noncompartmental pharmacokinetic parameters were determined from plasma and urine concentration-time data. RESULTS: PD 0200390 was rapidly absorbed; mean time to maximum plasma concentration was 1.66-3.24 h. Mean half-life in subjects with normal renal function was 5.36 h, and increased with worsening RI. Oral (CL/F) and renal (CL(R)) clearance rates decreased with deteriorating renal function, whereas area under the concentration-time curve (AUC(0-infinity)) values increased by 56, 117 and 436% in subjects with mild, moderate and severe RI, respectively, indicating increased PD 0200390 exposure. Regression analysis demonstrated that CL/F and CL(R) correlated with CLcr (r= 0.953 and 0.961, respectively). PD 0200390 was well tolerated in subjects with mild, moderate or no RI. The most common adverse events were somnolence, dizziness and headache; these occurred with greatest intensity in the severe RI group. CONCLUSIONS: PD 0200390 pharmacokinetic parameters (CL/F, CL(R) and AUC(0-infinity)) vary predictably with decreases in renal function; therefore dose adjustment may be required in individuals with RI.

Hipertensão arterial: classificação das principais drogas anti-hipertensivas / Arterial hypertension: main antihypertensive drugs classification

Os autores fazem um resumo das drogas anti-hipertensivas mais importantes, dando ênfase à classificação quanto ao mecanismo de sção, dosagem e principais efeitos colaterais inerentes a cada grupo, com algumas observações de valor. O presente trabalho visa dar uma visão panorâmica às drogas anti-hipertensivas, facilitando, em muito, sua compreensão.

[Calcium antagonists and ischemic heart disease]. / Kalcium-antagonisták és ischaemiás szívbetegség.

The authors give a short account about the significance, basic rules in regulation of intracellular (cytosolic) calcium homeostasis, distribution of calcium channels in different organs and characteristics of L channels in cardiovascular system. The different types of calcium channel blockers (dihydropyridines, phenylalkilamines, benzothiazepines), their similarities, dissimilarities (cardio, -vascular selectivity) are discussed. Differences in calcium homeostasis, in sympathetic innervation between vascular smooth- and heart muscle in briefly disputed. The basic role of endothel cell dysfunction in development of ischemic heart disease, the possible mechanisms (potential anti-remodeling, antifissuring, antiischemic, cardioprotective, sympaticolytic effects) of different calcium antagonists in the treatment of patients with ischemic heart disease are reviewed. Potential adverse effects of calcium channel blockers (proischemic, proarrhythmic, procongestive, prohaemorrhagic effects) are also mentioned. Finally the up-date preventive and therapeutic indications of different calcium channel blockers in the clinical management of patients with stable, unstable angina pectoris, silent myocardial ischemia, vasospastic angina, acute myocardial infarction is shortly reported.

Altered muscle calcium channel binding kinetics in autoimmune motoneuron disease.

While skeletal muscle is not apparently affected directly in amyotrophic lateral sclerosis (ALS), immunoglobulin G fractions purified from patients with ALS (ALS IgG) bind dihydropyridine (DHP)-sensitive L-type voltage-gated calcium channel (VGCC) antigen isolated from skeletal muscle in ELISA and Western immunoblot, and alter VGCC function in vitro. To determine whether muscle VGCC properties are altered in ALS, VGCC-enriched subsarcolemmal membrane fractions were prepared from biopsied quadriceps muscle of patients with ALS, with other neurologic diseases, or without apparent muscle disease, and tested for DHP binding with [3H]PN200-110. ALS muscle VGCCs possessed eightfold higher binding affinities for [3H]PN200-110 than did VGCCs from muscle fractions of most other patients, independent of denervation-induced increases in DHP binding site number. Similarly elevated DHP binding affinities were observed in specimens from patients with autoimmune motor neuropathies, suggesting that ALS and immune mediated motoneuron disease share skeletal muscle L-type VGCC alterations.

Sensory neuron N-type calcium currents are inhibited by both voltage-dependent and -independent mechanisms.

The voltage dependence of gamma-aminobutyric-acid- and norepinephrine-induced inhibition of N-type calcium current in cultured embryonic chick dorsal-root ganglion neurons was studied with whole-cell voltage-clamp recording. The inhibitory action of the neurotransmitters was comprised of at least two distinct modulatory components, which were separable on the basis of their differential voltage dependence. The first component, which we term "kinetic slowing", is associated with a slowing of the activation kinetics--an effect that subsides during a test pulse. The kinetic-slowing component is largely reversed at depolarized voltages (i.e., it is voltage-dependent). The second component, which we term "steady-state inhibition", is by definition not associated with a change in activation kinetics and is present throughout the duration of a test pulse. The steady-state inhibition is not reversed at depolarized voltages (i.e., it is voltage-independent). Although the two components can be separated on the basis of their voltage dependence, they appear to be indistinguishable in their time courses for onset and recovery as well as their rates of desensitization following multiple applications of transmitter. Furthermore, neither component requires cell dialysis, as both are observed using perforated-patch as well as whole-cell recording configurations. The co-existence in nerve terminals of both voltage-dependent and -independent mechanisms to modulate calcium channel function could offer a means of differentially controlling synaptic transmission under conditions of low- and high-frequency presynaptic discharge.