Caffeine inhibits inositol-trisphosphate-induced membrane potential oscillations in Xenopus oocytes.

Immature Xenopus oocytes injected with inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) give a complex electrophysiological response comprising an a early depolarizing spike followed by a burst of oscillations. These two components have been interpreted on the basis of an interaction between two internal calcium stores: an Ins(1,4,5) P3-sensitive pool responsible for the early spike which then primes an Ins(1,4,5) P3-insensitive pool to begin to oscillate through a process of calcium-induced calcium release (Berridge, M. J., J. Physiol., Lond. 403, 589-599 (1988)). The role of the latter was investigated in Xenopus oocytes by using the drug caffeine which can trigger calcium-induced calcium release in muscle cells. Caffeine had no effect on the early Ins(1,4,5)P3-induced spike but it suppressed the subsequent oscillations. The spontaneous oscillations observed in some oocytes were also abolished by caffeine. Oscillation amplitude and duration was slightly reduced following incubation of oocytes with adenosine or isobutylmethylxanthine. Because these two agents gave large membrane hyperpolarizations indicative of an increase in cyclic AMP, it can be concluded that this second messenger is not responsible for the inhibitory action of caffeine. The ability of caffeine to abolish oscillations while not affecting the early Ins(1,4,5) P3 response is discussed with regard to the two-pool model for generating calcium oscillations.     

Intrathecal bombesin is sympathoexcitatory and pressor in rat

Bombesin, a 14 amino-acid peptide, is pressor when administered intravenously in rat and pressor and sympathoexcitatory when applied intracerebroventricularly. To determine the spinal effects of bombesin, the peptide was administered acutely in the intrathecal space at around thoracic spinal cord level six of urethane-anesthetized, paralyzed, and bilaterally vagotomized rats. Blood pressure, heart rate, splanchnic sympathetic nerve activity (sSNA), phrenic nerve activity, and end-tidal CO(2) were monitored to evaluate changes in the cardiorespiratory systems. Bombesin elicited a long-lasting excitation of sSNA associated with an increase in blood pressure and tachycardia. There was a mean increase in arterial blood pressure of 52 ± 5 mmHg (300 μM; P < 0.01). Heart rate and sSNA also increased by 40 ± 4 beats/min (P < 0.01) and 162 ± 33% (P < 0.01), respectively. Phrenic nerve amplitude (PNamp, 73 ± 8%, P < 0.01) and phrenic expiratory period (+0.16 ± 0.02 s, P < 0.05) increased following 300 μM bombesin. The gain of the sympathetic baroreflex increased from -2.8 ± 0.7 to -5.4 ± 0.9% (P < 0.01), whereas the sSNA range was increased by 99 ± 26% (P < 0.01). During hyperoxic hypercapnia (10% CO(2) in O(2), 90 s), bombesin potentiated the responses in heart rate (-25 ± 5 beats/min, P < 0.01) and sSNA (+136 ± 29%, P < 0.001) but reduced PNamp (from 58 ± 6 to 39 ± 7%, P < 0.05). Finally, ICI-216,140 (1 mM), an in vivo antagonist for the bombesin receptor 2, attenuated the effects of 300 μM bombesin on blood pressure (21 ± 7 mmHg, P < 0.01). We conclude that bombesin is sympathoexcitatory at thoracic spinal segments. The effect on phrenic nerve activity may the result of spinobulbar pathways and activation of local motoneuronal pools.     

Chronic high cytosolic calcium decreases AICAR-induced AMPK activity via calcium/calmodulin activated protein kinase II signaling cascade

Calcium is important for muscle contraction and controls many cellular processes. Although there is evidence that calcium-mediated signals regulate AMP-activated protein kinase (AMPK) activity, the molecular mechanisms by which calcium regulates AMPK are poorly understood. To compare the function of sustained vs. intermittent calcium oscillations on AMPK activity and define specific signals in this pathway, we administered mice with aminoimidazole-carboxamide-ribonucleotide (AICAR) and caffeine with or without dantrolene. AMPK activity was increased by 10 d AICAR treatment (P < 0.01). Ten day caffeine treatment decreased AICAR-induced AMPK activity to control level. This repressed AMPK activity was blocked by dantrolene. Different calcium frequencies were simulated in C2C12 myotubes by alternating media containing caffeine and dantrolene. Intermittent calcium oscillation increased AMPK activity compared to control (P < 0.05), whereas sustained calcium oscillation decreases AICAR-induced AMPK activity to control level. This result suggests a biphasic control of AMPK activity by calcium. Knockdown of CaMKII expression by short-hairpin RNA resulted in increased AMPK phosphorylation by AICAR even in the presence of caffeine. These data show different calcium oscillations elicit distinct responses in muscle cells suggesting that the negative effects of chronic calcium treatment on AMPK activity is partly mediated through the CaMKII signals.     

Salt modulates vascular response through adenosine A2A receptor in eNOS-null mice: role of CYP450 epoxygenase and soluble epoxide hydrolase

High salt (HS) intake can change the arterial tone in mice, and the nitric oxide (NO) acts as a mediator to some of the receptors mediated vascular response. The main aim of this study was to explore the mechanism behind adenosine-induced vascular response in HS-fed eNOS(+/+) and eNOS(-/-) mice The modulation of vascular response by HS was examined using aortas from mice (eNOS(+/+) and eNOS(-/-)) fed 4% (HS) or 0.45% (NS) NaCl-diet through acetylcholine (ACh), NECA (adenosine-analog), CGS 21680 (A(2A) AR-agonist), MS-PPOH (CYP epoxygenase-blocker; 10(-5) M), AUDA (sEH-blocker; 10(-5) M), and DDMS (CYP4A-blocker; 10(-5) M). ACh-response was greater in HS-eNOS(+/+) (+59.3 ± 6.3%) versus NS-eNOS(+/+) (+33.3 ± 8.0%; P < 0.05). However, there was no response in both HS-eNOS(-/-) and NS-eNOS(-/-). NECA-response was greater in HS-eNOS(-/-) (+37.4 ± 3.2%) versus NS-eNOS(-/-) (+7.4.0 ± 3.8%; P < 0.05). CGS 21680-response was also greater in HS-eNOS(-/-) (+45.4 ± 5.2%) versus NS-eNOS(-/-)(+5.1 ± 5.0%; P < 0.05). In HS-eNOS(-/-), the CGS 21680-response was reduced by MS-PPOH (+7.3 ± 3.2%; P < 0.05). In NS-eNOS(-/-), the CGS 21680-response was increased by AUDA (+38.2 ± 3.3%; P < 0.05) and DDMS (+30.1 ± 4.1%; P < 0.05). Compared to NS, HS increased CYP2J2 in eNOS(+/+) (35%; P < 0.05) and eNOS(-/-) (61%; P < 0.05), but decreased sEH in eNOS(+/+) (74%; P < 0.05) and eNOS(-/-) (40%; P < 0.05). Similarly, CYP4A decreased in HS-eNOS(+/+) (35%; P < 0.05) and HS-eNOS(-/-) (34%; P < 0.05). These data suggest that NS causes reduced-vasodilation in both eNOS(+/+) and eNOS(-/-) via sEH and CYP4A. However, HS triggers possible A(2A)AR-induced relaxation through CYP epoxygenase in both eNOS(+/+) and eNOS(-/-).     

Involvement of the Endoplasmic Reticulum of Chromaffin Cells of the Rat Adrenal Gland in Calcium Signaling

We studied the involvement of the endoplasmic reticulum (ER) in calcium signaling in rat chromaffin cells. For this purpose, the following agents influencing the activity of the ER were used: (i) Caffeine that activates the release of Ca²⁺ from the endoplasmic store and (ii) thapsigargin that suppresses accumulation of calcium in the ER. The intracellular Ca²⁺ concentration was measured with the help of a calcium-sensitive dye, Fura-2AM, using the microfluorescent technique. Applications of caffeine led to a rise in the level of free Ca²⁺ in the cell cytosol and also to a decrease in the amplitude of calcium transients induced by depolarization of the plasma membrane under the action of a hyperpotassium solution. Under conditions of repeated caffeine applications, the amplitude of transients decreased to 9% of its initial value, which is explained by exhaustion of the calcium stores. The action of caffeine was restored when the calcium stores were re-filled under the action of depolarization of the plasma membrane. Thapsigargin completely removed the effect of caffeine and did not influence KCl-induced transients. Therefore, our experiments are indicative of a significant importance of the ER calcium stores for calcium signaling in chromaffin cells, which allows us to hypothesize that these stores play an important role in the control of secretion of catecholamines.     

Optical mapping of cryoinjured rat myocardium grafted with mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been shown to improve cardiac electrophysiology when administered in the setting of acute myocardial infarction. However, the electrophysiological phenotype of MSCs in situ is not clear. We hypothesize that MSCs delivered intramyocardially to cryoinjured myocardium can engraft, but will not actively generate, action potentials. Cryoinjury-induced scar was created on the left ventricular epicardial surface of adult rat hearts. Within 30 min, hearts were injected with saline (sham, n = 11) or bone marrow-derived MSCs (2 × 10(6)) labeled with 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine percholate (DiI; n = 16). At 3 wk, optical mapping and cell isolation were used to measure optical action potentials and calcium transients, respectively. Histological analysis confirmed subepicardial scar thickness and the presence of DiI-positive cells that express connexin-43. Optical action potential amplitude within the scar at MSC-positive sites (53.8 ± 14.3%) was larger compared with sites devoid of MSCs (35.3 ± 14.2%, P < 0.05) and sites within the scar of shams (33.5 ± 6.9%, P < 0.05). Evidence of simultaneous action potential upstroke, the loss of action potential activity following ablation of adjacent viable myocardium, and no rapid calcium transient response in isolated DiI+ cells suggest that the electrophysiological influence of engrafted MSCs is electrotonic. MSCs can engraft when directly injected into a cryoinjury and are associated with evidence of action potential activity. However, our results suggest that this activity is not due to generation of action potentials, but rather passive influence coupled from neighboring viable myocardium.     

Acylation-stimulating protein/C5L2-neutralizing antibodies alter triglyceride metabolism in vitro and in vivo

Acylation-stimulating protein (ASP), a lipogenic hormone, stimulates triglyceride (TG) synthesis and glucose transport upon activation of C5L2, a G protein-coupled receptor. ASP-deficient mice have reduced adipose tissue mass due to increased energy expenditure despite increased food intake. The objective of this study was to evaluate the blocking of ASP-C5L2 interaction via neutralizing antibodies (anti-ASP and anti-C5L2-L1 against C5L2 extracellular loop 1). In vitro, anti-ASP and anti-C5L2-L1 blocked ASP binding to C5L2 and efficiently inhibited ASP stimulation of TG synthesis and glucose transport. In vivo, neither anti-ASP nor anti-C5L2-L1 altered body weight, adipose tissue mass, food intake, or hormone levels (insulin, leptin, and adiponectin), but they did induce a significant delay in TG clearance [P < 0.0001, 2-way repeated-measures (RM) ANOVA] and NEFA clearance (P < 0.0001, 2-way RM ANOVA) after a fat load. After treatment with either anti-ASP or anti-C5L2-L1 antibody there was no change in adipose tissue AMPK activity, but neutralizing antibodies decreased perirenal TG mass (-38.4% anti-ASP, -18.8% anti-C5L2, P < 0.01-0.001) and perirenal LPL activity (-75.6% anti-ASP, -72.5% anti-C5L2, P < 0.05). In liver, anti-C5L2-L1 decreased TG mass (-42.8%, P < 0.05), whereas anti-ASP increased AMPK activity (+34.6%, P < 0.001). In the muscle, anti-C5L2-L1 significantly increased TG mass (+128.0%, P < 0.05), LPL activity (+226.1%, P < 0.001), and AMPK activity (+71.1%, P < 0.01). In addition, anti-ASP increased LPL activity (+164.4, P < 0.05) and AMPK activity (+53.9%, P < 0.05) in muscle. ASP/C5L2-neutralizing antibodies effectively block ASP-C5L2 interaction, altering lipid distribution and energy utilization.     

Caffeine-induced oscillations of the membrane potential inAplysia neurons

It has been found in culturedAplysia neurons, including L7 and L2–L6 neurons, that bath application of 40 mM caffeine evokes oscillations of the membrane potential (MP) with the amplitude of about 40 mV. The frequency of oscillations, on the crest of which action potentials (AP) arise, varied from 0.2 to 0.5 sec¹. The effect of caffeine was completely reversible. The MP waves demonstrated high sensitivity to membrane polarization: artificial depolarization increased the frequency of oscillations, while even subtle hyperpolarization resulted in a decrease in the frequency up to their complete disappearance. External application of CdCl₂ (1 mM), a nonspecific blocker of calcium channels, or ryanodine (50 μM, 20 min), release of Ca²⁻ from the intracellular stores, replacement of Ca²⁺ in the external medium by Mg²⁻, or Na⁺ by Li⁺, did not exert visible effect on the parameters of MP waves. It was concluded that Ca ions (changing of intracellular concentration of which is due to such processes as inward calcium current, ryanodine-sensitive caffeine-induced calcium release from the intracellular, stores, sodium-calcium exchange through the plasma membrane) do not play any significant part in generation of the MP waves. The most probable mechanism of caffeine-induced oscillations in the studied nerve cells is inhibition of voltage-activated outward potassium current and, as could be seen from our mathematical modeling, slowdown of inactivation of inward sodium current. It seems likely that these oscillations have a purely membrane origin. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 102–111, March–April, 2000.     

Identification of a physiological role for leptin in the regulation of ambulatory activity and wheel running in mice

Mechanisms regulating spontaneous physical activity remain poorly characterized despite evidence of influential genetic and acquired factors. We evaluated ambulatory activity and wheel running in leptin-deficient ob/ob mice and in wild-type mice rendered hypoleptinemic by fasting in both the presence and absence of subcutaneous leptin administration. In ob/ob mice, leptin treatment to plasma levels characteristic of wild-type mice acutely increased both ambulatory activity (by 4,000 ± 200 beam breaks/dark cycle, P < 0.05) and total energy expenditure (TEE; by 0.11 ± 0.01 kcal/h during the dark cycle, P < 0.05) in a dose-dependent manner and acutely increased wheel running (+350%, P < 0.05). Fasting potently increased ambulatory activity and wheel running in wild-type mice (AA: +25%, P < 0.05; wheel running: +80%, P < 0.05), and the effect of fasting was more pronounced in ob/ob mice (AA: +400%, P < 0.05; wheel running: +1,600%, P < 0.05). However, unlike what occurred in ad libitum-fed ob/ob mice, physiological leptin replacement attenuated or prevented fasting-induced increases of ambulatory activity and wheel running in both wild-type and ob/ob mice. Thus, plasma leptin is a physiological regulator of spontaneous physical activity, but the nature of leptin's effect on activity is dependent on food availability.     

Left ventricular systolic torsion correlates global cardiac performance during dyssynchrony and cardiac resynchronization therapy

Left ventricular (LV) systolic torsion is a primary mechanism contributing to stroke volume (SV). We hypothesized that change in LV torsion parallels changes in global systolic performance during dyssynchrony and cardiac resynchronization therapy (CRT). Seven anesthetized open chest dogs had LV pressure-volume relationship. Apical, basal, and mid-LV cross-sectional echocardiographic images were studied by speckle tracking analysis. Right atrial (RA) pacing served as control. Right ventricular (RV) pacing simulated left bundle branch block. Simultaneous RV-LV free wall and RV-LV apex pacing (CRTfw and CRTa, respectively) modeled CRT. Dyssynchrony was defined as the time difference in peak strain between earliest and latest segments. Torsion was calculated as the maximum difference between the apical and basal rotation. RA pacing had minimal dyssynchrony (52 ± 36 ms). RV pacing induced dyssynchrony (189 ± 61 ms, P < 0.05). CRTa decreased dyssynchrony (46 ± 36 ms, P < 0.05 vs. RV pacing), whereas CRTfw did not (110 ± 96 ms). Torsion during baseline RA was 6.6 ± 3.7°. RV pacing decreased torsion (5.1 ± 3.6°, P < 0.05 vs. control), and reduced SV, stroke work (SW), and dP/dt(max) compared with RA (21 ± 5 vs. 17 ± 5 ml, 252 ± 61 vs. 151 ± 64 mJ, and 2,063 ± 456 vs. 1,603 ± 424 mmHg/s, respectively, P < 0.05). CRTa improved torsion, SV, SW, and dP/dt(max) compared with RV pacing (7.7 ± 4.7°, 23 ± 3 ml, 240 ± 50 mJ, and 1,947 ± 647 mmHg/s, respectively, P < 0.05), whereas CRTfw did not (5.1 ± 3.6°, 18 ± 5 ml, 175 ± 48 mJ, and 1,699 ± 432 mmHg/s, respectively, P < 0.05). LV torsion changes covaried across conditions with SW (y = 0.94x+12.27, r = 0.81, P < 0.0001) and SV (y = 0.66x+0.91, r = 0.81, P < 0.0001). LV dyssynchrony changes did not correlate with SW or SV (r = -0.12, P = 0.61 and r = 0.08, P = 0.73, respectively). Thus, we conclude that LV torsion is primarily altered by dyssynchrony, and CRT that restores LV performance also restores torsion.     

Spontaneous myocardial calcium oscillations: overview with emphasis on ryanodine and caffeine

All mammalian cardiac preparations exhibit the capacity for periodic spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) (Ca2+ oscillations). The occurrence of such oscillations in unstimulated preparations and their periodicity depend on the species and the Ca2+ load on the cell. When the spontaneous frequency of these oscillations exceeds the rate of external simulation, they appear between stimulated contractions and impart a variable Ca2+-dependent component of diastolic tonus and a propensity for extrasystoles and arrhythmias to occur; these diastolic oscillations can also affect systolic function as well. Although enhancing the spontaneous frequency of Ca2+ release, caffeine depresses the oscillation amplitude, whereas ryanodine suppresses both frequency and amplitude. Detailed studies of oscillation characteristics and of the different effects of caffeine and ryanodine on them may provide an understanding of and may be useful for modeling SR Ca2+ uptake and release in intact preparations.     

Conduction and refractory disorders in the diabetic atrium

Diabetes mellitus (DM) is an independent risk of atrial fibrillation. However, its arrhythmogenic substrates remain unclear. This study sought to examine the precise propagation and the spatiotemporal dispersion of the action potential (AP) in the diabetic atrium. DM was induced by streptozotocin (65 mg/kg) in 8-wk-old male Wister rats. Optical mapping and histological analysis were performed in the right atrium (RA) from control (n = 26) and DM (n = 27) rats after 16 wk. Rate-dependent alterations of conduction velocity (CV) and its heterogeneity and the spatial distribution of AP were measured in RA using optical mapping. The duration of atrial tachyarrhythmia (AT) induced by rapid atrial stimulation was longer in DM (2.4 ± 0.6 vs. 0.9 ± 0.3 s, P < 0.05). CV was decreased, and its heterogeneity was greater in DM than control. Average action potential duration of 80% repolarization (APD(80)) at pacing cycle length (PCL) of 200 ms from four areas within the RA was prolonged (53 ± 2 vs. 40 ± 3 ms, P < 0.01), and the coefficient of variation of APD(80) was greater in DM than control (0.20 ± 0.02 vs. 0.15 ± 0.01%, P < 0.05). The ratio of APD(80) at PCL shorter than 200 ms to that at 200 ms was smaller (P < 0.001), and the incidence of APD alternans was higher in DM than control (100 vs. 0%, P < 0.001). Interstitial fibrosis was greater and connexin 40 expression was lower in DM than control. The remodeling of the diabetic atrium was characterized as follows: greater vulnerability to AT, increased conduction slowing and its heterogeneity, the prolongation of APD, the increase in spatial dispersion and frequency-dependent shortening of APD, and increased incidence of APD alternans.     

Purification and characterization of a novel caffeine oxidase from Alcaligenes species

Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The optimal medium for caffeine oxidase production by this strain was (w/v) NaNO(3), 0.4%; KH(2)PO(4), 0.15%; Na(2)HPO(4), 0.05%; FeCl(3).6H(2)O, 0.0005%; CaCl(2).2H(2)O, 0.001%; MgSO(4).7H(2)O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life of 20 min at 50 degrees C had maximal activity at pH 7.5 and 35 degrees C. The purified caffeine oxidase had strict substrate specificity towards caffeine (K(m) 8.94 microM and V(max) 47.62 U mg protein(-1)) and was not able to oxidize xanthine and hypoxanthine. The enzyme activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca(+2), Mg(+2) and Na(+), but was completely inhibited by Co(+2), Cu(+2) and Mn(+2) at 1mM level. The novel caffeine oxidase isolated here from Alcaligenes species CF8 may be useful in biotechnological processes including waste treatment and biosensor development.     

Synchronization of ventricular fibrillation with real-time feedback pacing: implication to low-energy defibrillation

Wavefront synchronization is an important aspect preceding the termination of ventricular fibrillation (VF). We evaluated the defibrillation efficacy of a novel multisite pacing algorithm using optical recording-guided synchronized pacing (SyncP) in the excitable gaps. We compared the effects of SyncP with traditional overdrive pacing (ODP) at 90% of the VF cycle length (VFCL) and high-frequency pacing (HFP; 43-215 Hz) on spontaneous VF termination in isolated rabbit hearts. For SyncP, the pacing current was triggered by the activation of a reference site and was delivered when the optical potential of the pacing site was in an excitable gap. We measured VFCL and the spatial dispersion of VFCL (SDCL) from five points (3 points in the paced area and 2 points in the nonpaced area) and the distribution of phase singularities during the prepacing, pacing, and postpacing periods. The results showed that 1) the VF termination rate of SyncP (16.0%, n = 106) was higher than that of ODP (2.1%, n = 48, P < 0.01) or HFP (1.6%, n = 129, P < 0.0001); 2) energy consumption for SyncP (7.6 +/- 9.3 mJ) was significantly lower than that of ODP (14.0 +/- 14.8 mJ, P < 0.0001); and 3) SyncP, but not ODP or HFP, decreased SDCL in the paced area during the pacing (P < 0.01) and postpacing (P < 0.05) periods compared with the prepacing period. We conclude that SyncP is effective in inducing wavefront synchronization and is more effective at facilitating spontaneous VF termination than non-SyncP.     

Loss of responsiveness of circular smooth muscle cells from the guinea pig ileum is associated with changes in gap junction coupling

Gap junction coupling and neuromuscular transmission to smooth muscle were studied in the first 4 h after preparations were set up in vitro. Intracellular recordings were made from smooth muscle cells of guinea pig ileum. Fast inhibitory junction potentials (IJPs) were small (1.3 ± 1.0 mV) in the first 30 min but increased significantly over the first 120 min to 15.8 ± 0.9 mV (n = 12, P < 0.001). Comparable increases in slow IJPs and excitatory junction potentials were also observed. During the same period, resting membrane potential depolarized from -58.8 ± 1.4 to -47.2 ± 0.4 mV (n = 12, P < 0.001). Input resistance, estimated by intracellular current injection, decreased in parallel (P < 0.05), and dye coupling, measured by intracellular injection of carboxyfluorescein, increased (P < 0.001). Input resistance was higher and dye coupling was less in longitudinal than circular smooth muscle cells. Gap junction blockers [carbenoxolone (100 μM), 18β-glycyrrhetinic acid (10 μM), and 2-aminoethoxydiphenyl borate (50 μM)] hyperpolarized coupled circular smooth muscle cells, reduced the amplitude of fast and slow IJPs and excitatory junction potentials, increased input resistance, and reduced dye coupling. Local application of ATP (10 mM) mimicked IJPs and showed comparable increases in amplitude over the first 120 min; carbenoxolone and 2-aminoethoxydiphenyl borate significantly reduced ATP-evoked hyperpolarizations in coupled cells. In contrast, synaptic transmission between myenteric neurons was not suppressed during the first 30 min. Gap junction coupling between circular smooth muscle cells in isolated preparations was initially disrupted but recovered over the next 120 min to a steady level. This was associated with potent effects on neuromuscular transmission and responses to exogenous ATP.     

Caffeine intake improves intense intermittent exercise performance and reduces muscle interstitial potassium accumulation

The effect of oral caffeine ingestion on intense intermittent exercise performance and muscle interstitial ion concentrations was examined. The study consists of two studies (S1 and S2). In S1, 12 subjects completed the Yo-Yo intermittent recovery level 2 (Yo-Yo IR2) test with prior caffeine (6 mg/kg body wt; CAF) or placebo (PLA) intake. In S2, 6 subjects performed one low-intensity (20 W) and three intense (50 W) 3-min (separated by 5 min) one-legged knee-extension exercise bouts with (CAF) and without (CON) prior caffeine supplementation for determination of muscle interstitial K(+) and Na(+) with microdialysis. In S1 Yo-Yo IR2 performance was 16% better (P < 0.05) in CAF compared with PLA. In CAF, plasma K(+) at the end of the Yo-Yo IR2 test was 5.2 ± 0.1 mmol/l with no difference between the trials. Plasma free fatty acids (FFA) were higher (P < 0.05) in CAF than PLA at rest and remained higher (P < 0.05) during exercise. Peak blood glucose (8.0 ± 0.6 vs. 6.2 ± 0.4 mmol/l) and plasma NH(3) (137.2 ± 10.8 vs. 113.4 ± 13.3 μmol/l) were also higher (P < 0.05) in CAF compared with PLA. In S2 interstitial K(+) was 5.5 ± 0.3, 5.7 ± 0.3, 5.8 ± 0.5, and 5.5 ± 0.3 mmol/l at the end of the 20-W and three 50-W periods, respectively, in CAF, which were lower (P < 0.001) than in CON (7.0 ± 0.6, 7.5 ± 0.7, 7.5 ± 0.4, and 7.0 ± 0.6 mmol/l, respectively). No differences in interstitial Na(+) were observed between CAF and CON. In conclusion, caffeine intake enhances fatigue resistance and reduces muscle interstitial K(+) during intense intermittent exercise.     

Sympathetic Activation Does Not Affect the Cardiac and Respiratory Contribution to the Relationship between Blood Pressure and Pial Artery Pulsation Oscillations in Healthy Subjects

Introduction

Using a novel method called near-infrared transillumination backscattering sounding (NIR-T/BSS) that allows for the non-invasive measurement of pial artery pulsation (cc-TQ) and subarachnoid width (sas-TQ) in humans, we assessed the influence of sympathetic activation on the cardiac and respiratory contribution to blood pressure (BP) cc-TQ oscillations in healthy subjects.

Methods

The pial artery and subarachnoid width response to handgrip (HGT) and cold test (CT) were studied in 20 healthy subjects. The cc-TQ and sas-TQ were measured using NIR-T/BSS; cerebral blood flow velocity (CBFV) was measured using Doppler ultrasound of the left internal carotid artery; heart rate (HR) and beat-to-beat mean BP were recorded using a continuous finger-pulse photoplethysmography; respiratory rate (RR), minute ventilation (MV), end-tidal CO₂ (EtCO2) and end-tidal O₂ (EtO2) were measured using a metabolic and spirometry module of the medical monitoring system. Wavelet transform analysis was used to assess the relationship between BP and cc-TQ oscillations.

Results

HGT evoked an increase in BP (+15.9%; P<0.001), HR (14.7; P<0.001), SaO₂ (+0.5; P<0.001) EtO₂ (+2.1; P<0.05) RR (+9.2%; P = 0.05) and MV (+15.5%; P<0.001), while sas-TQ was diminished (-8.12%; P<0.001), and a clear trend toward cc-TQ decline was observed (-11.0%; NS). CBFV (+2.9%; NS) and EtCO₂ (-0.7; NS) did not change during HGT. CT evoked an increase in BP (+7.4%; P<0.001), sas-TQ (+3.5%; P<0.05) and SaO₂(+0.3%; P<0.05). HR (+2.3%; NS), CBFV (+2.0%; NS), EtO₂ (-0.7%; NS) and EtCO₂ (+0.9%; NS) remained unchanged. A trend toward decreased cc-TQ was observed (-5.1%; NS). The sas-TQ response was biphasic with elevation during the first 40 seconds (+8.8% vs. baseline; P<0.001) and subsequent decline (+4.1% vs. baseline; P<0.05). No change with respect to wavelet coherence and wavelet phase coherence was found between the BP and cc-TQ oscillations.

Conclusions

Short sympathetic activation does not affect the cardiac and respiratory contribution to the relationship between BP—cc-TQ oscillations. HGT and CT display divergent effects on the width of the subarachnoid space, an indirect marker of changes in intracranial pressure.     

Functional Modeling of the Shift in Cellular Calcium Dynamics at the Onset of Synchronization in Smooth Muscle Cells

In the present paper we address the nature of synchronization properties found in populations of mesenteric artery smooth muscle cells. We present a minimal model of the onset of synchronization in the individual smooth muscle cell that is manifested as a transition from calcium waves to whole-cell calcium oscillations. We discuss how different types of ion currents may influence both amplitude and frequency in the regime of whole-cell oscillations. The model may also explain the occurrence of mixed-mode oscillations and chaotic oscillations frequently observed in the experimental system.     

Differential regulation of sympathetic burst frequency and amplitude following acute hypoxia in humans

Current evidence suggests that the persistent sympathetic nerve activity (SNA), commonly observed after exposure to hypoxia (HX), is mediated by chemoreceptor sensitization and or baroreflex resetting. Evidence in humans and animals suggests that these reflexes may independently regulate the frequency (gating) and amplitude (neuronal recruitment) of SNA bursts. In humans (n = 7), we examined the regulation of SNA following acute isocapnic HX (5 min; end-tidal Po(2) = 45 Torr) and euoxic hypercapnia (HC; 5 min; end-tidal Pco(2) = +10 from baseline). HX increased SNA burst frequency (21 ± 7 to 28 ± 8 bursts/min, P < 0.05) and amplitude (99 ± 10 to 125 ± 19 au, P < 0.05) as did HC (14 ± 6 to 22 ± 10 bursts/min, P < 0.05 and 100 ± 12 to 133 ± 29 au, P < 0.05, respectively). Burst frequency (26 ± 7 bursts/min, P < 0.05), but not amplitude (97 ± 12 au), remained elevated 10 min post-HX. The change in burst amplitude (but not frequency) was significantly related to the measured change in ventilation (r(2) = 0.527, P < 0.001). Both frequency and amplitude decreased during recovery following HC. These data indicate the differential regulation of pattern and magnitude of sympathetic outflow in humans with sympathetic persistence following HX being specific to burst frequency and not amplitude.     

L-type Ca2+ channels and K+ channels specifically modulate the frequency and amplitude of spontaneous Ca2+ oscillations and have distinct roles in prolactin release in GH3 cells

GH3 cells showed spontaneous rhythmic oscillations in intracellular calcium concentration ([Ca2+]i) and spontaneous prolactin release. The L-type Ca2+ channel inhibitor nimodipine reduced the frequency of Ca2+ oscillations at lower concentrations (100nM-1 microM), whereas at higher concentrations (10 microM), it completely abolished them. Ca2+ oscillations persisted following exposure to thapsigargin, indicating that inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores were not required for spontaneous activity. The K+ channel inhibitors Ba2+, Cs+, and tetraethylammonium (TEA) had distinct effects on different K+ currents, as well as on Ca2+ oscillations and prolactin release. Cs+ inhibited the inward rectifier K+ current (KIR) and increased the frequency of Ca2+ oscillations. TEA inhibited outward K+ currents activated at voltages above -40 mV (grouped within the category of Ca2+ and voltage-activated currents, KCa,V) and increased the amplitude of Ca2+ oscillations. Ba2+ inhibited both KIR and KCa,V and increased both the amplitude and the frequency of Ca2+ oscillations. Prolactin release was increased by Ba2+ and Cs+ but not by TEA. These results indicate that L-type Ca2+ channels and KIR channels modulate the frequency of Ca2+ oscillations and prolactin release, whereas TEA-sensitive KCa,V channels modulate the amplitude of Ca2+ oscillations without altering prolactin release. Differential regulation of these channels can produce frequency or amplitude modulation of calcium signaling that stimulates specific pituitary cell functions.