Paired-Pulse Facilitation of Transmitter Release at Different Levels of Extracellular Calcium Concentration

High-frequency synaptic activity can cause facilitation of transmitter release due to accumulation of “residual Ca²⁺” at the nerve terminal. However, the mechanism of this phenomenon is still under debate. Here we show that, using extracellular recording from frog cutaneous pectoris muscle, paired-pulse facilitation (PPF) at the frog neuro-muscular junction decays in two or three-exponential manner depending upon the extracellular Ca²⁺ concentration ([Ca²⁺]_e). First, second and “early” PPF components are analyzed and described in this study. Considering the dependence of PPF on [Ca²⁺]_e, existence of several specific high-affinity intra-terminal Ca²⁺-binding sites that underlie the facilitation of transmitter release at the frog neuro-muscular junction is proposed.     

On the value of seasonal mammals for identifying mechanisms underlying the control of food intake and body weight

This article is part of a Special Issue “Energy Balance”.     

Sequence, cloning, expression and characterisation of the 81-kDa surface membrane protein (P80) of Mycoplasma agalactiae

In response to heat-stable enterotoxin of Vibrio cholerae non-O1, the initial rise of cytosolic Ca(2+) occurred with activation of IP(3). Chelation of extracellular Ca(2+) with EGTA and suspension of cells in Ca(2+) free buffer both demonstrated the involvement of internal stores in the rise of [Ca(2+)]i. Cells pretreated with dantrolene resulted in decrease of [Ca(2+)]i response which suggested that the rise of intracellular level of Ca(2+) was mostly due to the mobilization from IP(3) sensitive stores. When the cytosolic Ca(2+) was chelated by loading the cells with BAPTA, NAG-ST could not induce Ca(2+) entry to the cell as assessed by Mn(2+) quenching of fura-2 fluorescence which suggested that calcium influx across the plasma membrane depends upon initial rise of this bivalent cation that maintained the sustained phase of [Ca(2+)]i response. Addition of toxin to the fura-2-loaded cells, preincubated with lanthanum chloride, resulted in reduction of [Ca(2+)]i level with a short duration of irregular sustained phase further suggesting that the influx of Ca(2+) across the plasma membrane might be through the calcium channel.     

On the role of Ca2+-calmodulin-dependent and cAMP-dependent protein phosphorylation in the circadian rhythm ofNeurospora crassa

Summary Pulses of some Ca²⁺ channel blockers (dantrolene, Co²⁺, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5–9 h) phase shifts of the circadian conidiation rhythm ofNeurospora crassa. Pulses of high Ca²⁺, or of low Ca²⁺, a Ca²⁺ ionophore (A23187) together with Ca²⁺, and other Ca²⁺ channel blockers (La³⁺, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca²⁺ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca²⁺ (Cornelius et al., 1989).Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6–9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180° out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0–12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0.Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with³⁵S-thio -ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca²⁺ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42° C) temperatures.Altogether, the results indicate that Ca²⁺-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism ofNeurospora. Ca²⁺ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism.     

In vivo activation of cirral movement in Stylonychia by calcium

Summary Motor responses of cirri (= organelles consisting of bundles of cilia) in the protozoan Stylonychia are elicited by positive or negative shifts of the membrane voltage from its resting state. The same responses are evoked at voltages near the Ca²⁺ equilibrium potential (E_Ca) applying extremely positive steps under voltage clamp. Motor responses recorded at large positive voltages approaching E_Ca from the negative side corresponded to cirral activation following physiological depolarization from the resting potential (DCA). The hyperpolarization-induced activation of the cirri (HCA) was documented during step potentials positive to E_Ca, suggesting that the observed HCA of the cirri resulted from an efflux of Ca²⁺ from the ciliary space as compared with DCA, which is related to Ca²⁺ influx. The ciliary responses were graded functions of the rising outward or inward driving force for Ca²⁺. Slopes of reciprocal plots of response latencies near E_Ca as a function of membrane potential indicate a removal of Ca²⁺ during HCA which exceeds the free intraciliary Ca²⁺ content at rest. It is suggested that this excess Ca²⁺ is released from axonemal binding sites.     

Presynaptic Mechanism of Action of 4-Aminopyridine: Changes in Intracellular Free Ca2+ Concentration and Its Relationship to B-50 (GAP-43) Phosphorylation

Abstract: Recently we have shown that 4-aminopyridine (4-AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca²⁺. Hence, we were interested in the relationship between changes induced by 4-AP in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and B-50 phosphorylation in synaptosomes. 4-AP (100 μ M ) elevates the [Ca²⁺]_i (as determined with fura-2) to approximately the same extent as depolarization with 30 m M K⁺ (from an initial resting level of 240 n M to ∼480 n M after treatment). However, the underlying mechanisms appear to be different: In the presence of 4-AP, depolarization with K⁺ still evoked an increase in [Ca²⁺]_i, which was additive to the elevation caused by 4-AP. Several Ca²⁺ channel antagonists (CdCl₂, LaCl₃, and diphenylhydantoin) inhibited the increase in B-50 phosphorylation by 4-AP. It is interesting that the increase in [Ca²⁺]_i and the increase in B-50 phosphorylation by 4-AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na⁺ channels in this action. These results suggest that 4-AP (indirectly) stimulates both Ca²⁺ influx and B-50 phosphorylation through voltage-dependent channels by a mechanism dependent on Na⁺ channel activity.     

Antihypertensive medications and survival in patients with cancer: A population-based retrospective cohort study

Background: The association between antihypertensive medications and survival in cancer patients remains unclear. Objectives: To explore the association between classes of antihypertensive drugs and survival in cancer patients. Methods: Provincial Cancer Registry data was linked with a Provincial Drug Program Information Network (DPIN) for patients with lung (n = 4241), colorectal (n = 3967), breast (n = 4019) or prostate (n = 3355) cancer between the years of 2004 and 2008. Cox regression analyses were used to compare survival of patients using beta blockers (BBs), angiotensin-converting enzyme inhibitors/receptor blockers (ACEi/ARB), calcium channel blockers (CCBs) or thiazide diuretics (TDs) to survival of patients who did not use any of these antihypertensive drugs. Survival of patients using only one class of antihypertensive drugs were compared to each other, with BBs as the reference class. Results: Compared to the antihypertensive drug non-user cohort, BBs had no effect on survival for any of the cancers. ACEi/ARBs use was weakly associated with increased deaths for breast cancer (HR: 1.22, 95% CI: 1.04–1.44) and lung cancer (HR: 1.11, 95% CI: 1.03–1.21) patients. Deaths were also increased with CCB use in patients with breast cancer (HR: 1.22, 95% CI: 1.02–1.47) and with TD use in lung cancer patients (HR: 1.1, 95% CI: 1.01–1.19). There was strong evidence (p-value <0.0001) of an increase in deaths with TD use for colorectal (HR: 1.28, 95% CI: 1.15–1.42), and prostate (HR 1.41, 1.2–1.65) cancer patients. When including only antihypertensive drug users prescribed one drug class, lung cancer patients receiving CCBs had improved survival compared to BBs (HR 0.79, 95% CI: 0.64–0.98). Conclusions: Some classes of antihypertensive agents are associated with a decreased survival in certain cancers. The decrease could be due to more comorbidities in antihypertensive drug users. However, CCB use was associated with improved survival in lung cancer patients.     

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1^−/−) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca²⁺ cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1^−/− compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca²⁺ transient (Δ[Ca²⁺]_i) responses in NOS1^−/− cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca²⁺]_i across the range of pacing frequencies and increased myofilament Ca²⁺ sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca²⁺ reuptake, and restored SR Ca²⁺ content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca²⁺ leak, HYD improves Ca²⁺ cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.     

Influence of weaning regimen on intake,growth characteristics and plasma blood metabolites in male buffalo calves

Experiment was conducted to evaluate the effect of weaning age on growth performance, feed intake, feed efficiency (FE) and blood metabolites in Nili-Ravi male buffalo (Bubalus bubalis) calves. Twenty-four male buffalo calves were assigned to one of the three treatment groups: continuous milk feeding (CMF), limited milk feeding (LMF) and early weaning (EW), and weaned off milk at 12, 10 and 8 weeks of age, respectively. For the first 3 days after birth, calves in all three treatments were fed colostrum, and were then moved to individual milk feeding at 10% of BW for the next 6 weeks. Thereafter, the provision of milk to the CMF group was gradually tapered to zero through week 12, using week 6 intakes as a base. The LMF calves were fed milk at 7.5%, 5.0%, 3.5%, and 1.5% of BW during weeks 7 to 10, respectively. Lastly, calves in the EW group were fed milk at 5.0% and 2.5% of BW at weeks 7 and 8, respectively. Calf starter (CS) feed was also provided ad libitum from weeks 2 to 12 and individual intakes were recorded on a daily basis. Blood samples were taken from weeks 6 to 12, on a weekly basis; whereas, the BW, heart girth, withers height and hip width were measured at the start of experiment and later on a weekly basis. Weight gain, average daily gain, and body measurements were the same across all three groups. Milk intake was lower (P < 0.05), whereas CS intake was greater (P < 0.05) in the EW calves compared with the other treatment groups. Dry matter intake was greater (P < 0.05) in the EW and LMF calves compared with the CMF calves. The FE was greater (P < 0.05) in the CMF calves compared with the LMF and EW treatment groups. Blood glucose concentration was similar among the treatments; however, blood urea nitrogen was greater (P < 0.05) in the EW calves compared with the CMF and LMF groups. Plasma concentration of non-esterified fatty acids was higher (P < 0.05) in the EW calves compared with the CMF calves. In light of these results, it is evident that buffalo calves can be successfully weaned as early as 8 weeks of age without negatively affecting their growth performance.     

Circadian rhythm in hypothalamic leptin receptor (Ob-Rb) mRNA expressions and cerebrospinal fluid and circulating glucose and leptin levels in lactating rats

In lactating animals, the food consumption increases several-fold for milk supply to the pups. The present study was conducted to clarify the relationship between the hyperphagia during lactation and hypothalamic leptin receptor (Ob-Rb) mRNA expression, cerebrospinal fluid (CSF) and circulating leptin and glucose levels. Food intakes significantly higher in lactation than in non-lactation at all time points (3 points: light phase, 4 points: dark phase) of the day. However, the expression of the hypothalamic Ob-Rb mRNA showed similar circadian rhythms in both the non-lactation and lactation, with only slight differences between the two groups. CSF leptin and glucose levels were constant throughout the day in both non-lactation and lactation, and there was almost no difference between the two groups at each time point. Circulating leptin and glucose levels showed circadian rhythms only in the non-lactating period, and were lower in lactation than in non-lactation, especially in the dark phase. In conclusion, the present study provides evidence that Ob-Rb mRNA expression fluctuates in the lactation period as well as in the non-lactation period, suggesting that the expression profile of whole hypothalamic Ob-Rb may not contribute to the difference in food consumption between lactation and non-lactation, and that chronic decrease in blood glucose levels may be associated with the increase in food consumption during lactation.