[Evaluation of a pilot health promotion and stress management program for Pharmacy and Biochemistry students and professionals]. / Evaluación de un programa piloto de promoción de salud y prevención del estrés dirigido a estudiantes y profesionales de las carreras de Farmacia y Bioquímica.

OBJECTIVES: The beneficial results of a theory-practice pilot stress management program for Pharmacy and Biochemistry professionals and students. Its importance as a complement of traditional academic education, as well as its potential for Pharmaceutical Care is also discussed. MATERIALS AND METHODS: A total of 27 students and 26 professionals took part in a program of 10 sessions, aimed at improving stress management. Ten of the students and 10 professionals were randomly assigned to control groups. Salivary cortisol levels and anxiety level tests before and after the program were used to assess efficacy. RESULTS: Both the cortisol and the anxiety levels significantly decreased among students and professionals after the program, whereas it significantly increased in the student control group. Anxiety levels significantly decreased in both students and professionals. CONCLUSIONS: This type of pilot program proved effective for students. In the case of health professionals, the sample size needs to be increased in order to achieve an acceptable level of statistical power. Considering the shift of the pharmaceutical profession towards Pharmaceutical Care, the training of competences and attitudes like those described in this work could be of value.

Agonist-like activity of antibodies directed against the second extracellular loop of the human cardiac serotonin 5-HT4(e) receptor in transfected COS-7 cells.

We have previously reported that antipeptide antibodies directed against the second extracellular loop of the cardiac h5-HT4 receptor could block the activation of the L-type Ca channel in human atrial cardiomyocytes. In this paper we investigate the immunological and physiological activity of these antibodies, in a cell system expressing a larger amount of receptors than the atrial cells. The recombinant receptor was expressed at the surface of COS-7 cells under an active form (serotonin, EC50 = 1.81 x 10(-7) M), at a high level (375 +/- 25 fmol receptor/mg total protein) and was able to bind a specific ligand (GR113808) with a high affinity (Kd = 0.28 +/- 0.05 nM). In this system, the same anti-peptide antibodies used for the cardiac cells induced an "agonist-like" effect on the recombinant h5-HT4 receptor. These results are in line with those shown for others G-protein coupled receptors, as adrenoreceptors. In addition, this work showed that the effect of the antibodies is not only dependent on the epitopic region recognised but also on the molecular density and/or the cellular environment of the target receptors. Finally, our results support the hypothesis that the h5-HT4 receptor could be a new target for autoantibodies in patients with atrial arrhythmia.

cGMP-mediated inhibition of cardiac L-type Ca(2+) current by a monoclonal antibody against the M(2) ACh receptor.

The effects of a monoclonal antibody (B8E5) directed against the second extracellular loop of the muscarinic M(2) receptor were studied on the L-type Ca(2+) currents (I(Ca,L)) of guinea pig ventricular myocytes using the whole cell patch-clamp technique. Similar to carbachol, B8E5 reduced the isoproterenol (ISO)-stimulated I(Ca,L) but did not significantly affect basal I(Ca,L). Atropine blocked the inhibitory effect of B8E5. The electrophysiological parameters of ISO-stimulated I(Ca,L) were not modified in presence of B8E5. Inhibition of I(Ca,L) by B8E5 was still observed when intracellular cAMP was either enhanced by forskolin or maintained constant by using a hydrolysis-resistant cAMP analog (8-bromoadenosine 3',5'-cyclic monophosphate) or by applying the phosphodiesterase inhibitor IBMX. The effect of B8E5 was mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate, a potent stimulator of cGMP-dependent protein kinase, and prevented by a selective inhibitor of nitric oxide-sensitive guanylyl cyclase [1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one]. These results indicate that the antibody B8E5 inhibits the beta-adrenergic-stimulated I(Ca,L) through activation of the M(2) muscarinic receptor and further suggest that the antibody acts not via the classical pathway of decreasing intracellular cAMP, but rather by increasing cGMP.

Inhibitory activity of antibodies against the human atrial 5-HT(4)receptor.

Antibodies directed against the second extracellular loop of G protein-coupled receptors have been shown to exert "agonist-like" activities. In order to test the hypothesis that this is a general phenomenon, antibodies were raised in rabbits against a synthetic peptide corresponding to the second extracellular loop of the newly sequenced human cardiac 5-HT(4)receptor. The antibodies were affinity-purified and shown to recognize the 5-HT(4)receptor in immunoblots of Chinese hamster ovary (CHO) cells expressing the receptor. The antibodies had no intrinsic effect but could depress the activation of L -type calcium channel induced by serotonin in human atrial cells. This antagonist-like effect was exerted both by intact IgG and by Fab fragments. These results are physiologically important since it has been shown that the 5-HT(4)receptor could be a target for autoantibodies in mothers at risk of giving birth to children with neonatal atrio-ventricular block.

Anti-SSA/Ro52 autoantibodies blocking the cardiac 5-HT4 serotoninergic receptor could explain neonatal lupus congenital heart block.

The 52-kDa SSA/Ro (Ro52) ribonucleoprotein is an antigenic target strongly associated with the autoimmune response in mothers whose children develop neonatal lupus and congenital heart block. When sera from patients with systemic lupus erythematosus were used as autoimmune controls in an enzyme immunoassay to screen for antibodies against the human serotoninergic 5-HT4-receptor, a high correlation was found between the presence of anti-Ro52 protein antibodies in such sera and antibodies reacting with a synthetic peptide, corresponding to the second extracellular loop of the human 5-HT4 receptor (amino acid residues 165-185). Homology scanning between the 5-HT4 peptide and the sequence of the Ro52 protein indicated two potential common epitopes located between residues 365 and 396 of the Ro52 protein. Cross-reactivity was found between the peptide derived from the 5-HT4 receptor, and a peptide corresponding to residues 365-382 of the Ro52 protein. Autoantibodies, affinity-purified on the 5-HT4 receptor peptide, specifically recognized both the Ro52 protein and the 5-HT4 receptor protein in immunoblots. The affinity-purified antibodies antagonized the serotonin-induced L-type Ca channel activation on human atrial cells. This effect could explain the electrophysiological abnormalities in neonatal lupus.

Calcium-dependent modulation of the plateau phase of action potential in isolated ventricular cells of rabbit heart.

[Ca2+]i-dependent modulation of the action potential has been studied in Fura-2 dialysed ventricular myocytes of the rabbit using the whole-cell current-clamp method. Fifteen consecutive action potentials (AP1-AP15) and [Ca2+]i transients were elicited at a frequency of 0.2 Hz. A single, brief application of caffeine (during AP9) first enhanced and thereafter attenuated the [Ca2+]i transients accompanying AP9 and AP10-AP12, respectively. This approach provided direct comparison between time courses of action potentials: during the initial steady state (e.g. AP8) and when Ca2+ release from the sarcoplasmic reticulum was increased by caffeine (AP9) or decreased by depletion (AP10). The increase in [Ca2+]i facilitated repolarization and decreased action potential duration. However, action potentials at reduced Ca2+ release (AP10) had longer duration than during steady state. The caffeine-induced changes in L-type Ca2+ current (ICa,L), during voltage-clamp conditions partially explained the effects of caffeine on action potentials. When ICa,L was blocked by 500 micromol L-1 Cd2+, enhanced [Ca2+]i transients revealed an extra current component which was outward at +10 mV and inward at the resting membrane potential (most probably the transient inward current). In the presence of Cd2+, however, AP8 and AP10 had identical time courses, suggesting that ICa,L alone was responsible for the lengthening of AP10. Alterations in the transmembrane Na+ gradient resulted in changes of the steady state action potential durations (AP8) consistently with the expected modulation of the Na+-Ca2+ exchange current. However, the contribution of this current to the [Ca2+]i-dependent behaviour of action potential plateau could not be demonstrated.

Block of gating currents related to K+ channels as a mechanism of action of clofilium and d-sotalol in isolated guinea-pig ventricular heart cells.

1 The possibility that the class III antiarrhythmic drugs clofilium and d-sotalol might affect delayed rectifier potassium channels at the level of their gating currents was assessed with the whole-cell patch-clamp technique in guinea-pig isolated ventricular heart cells. 2 Clofilium (up to 20 microM) and d-sotalol (1 microM) did not decrease the Na current, the L-type Ca current or the background K current iKl, but significantly depressed the time-dependent delayed outward K current iK. 3 Clofilium partially decreased in a dose-dependent manner (1-20 microM) QON of intramembrane charge movements (ICM) elicited by a depolarizing pulse applied from a holding potential of -110 mV or following a 100 ms inactivating prepulse to -50 mV. D-sotalol (1 microM) also decreased QON. Channel density estimated from the clofilium-sensitive ICM closely matched that of the delayed rectifier channels. 4 Clofilium and d-sotalol decreased QOFF seen on repolarization in a dose- and voltage-dependent manner. The kinetics of the decay of the OFF gating currents were not affected, and only the fast phase was depressed. 5 In control conditions, QON availability with voltage was most of the time well described by two inactivating components. In the presence of clofilium and d-sotalol, a complex behaviour of QON availability was observed, unmasking additional components. The reactivation kinetics of QON after a 500 ms inactivating pulse to 0 mV was not affected. 6 We conclude that delayed rectifier K channels significantly contribute to QON and QOFF of ICM in guinea-pig ventricular heart cells, besides Na and Ca channels, and that clofilium and d-sotalol directly interact with these K channels proteins by affecting their gating properties.

An agonist-like monoclonal antibody against the human beta2-adrenoceptor.

Monoclonal antibodies were produced against a peptide corresponding to the second extracellular loop of the human beta2-adrenoceptor. One of these monoclonals, inducing an agonist-like effect in neonatal rat cardiomyocytes, was used to define the structural and physiological basis of this activity. The epitope recognized by the antibody corresponds to the sequence Trp-Tyr-Arg-Ala-Thr-His-Gln-Glu as determined by peptide scanning. Analysis by alanine modification of the peptide epitope showed the importance of the Trp, and Glu residues in antibody recognition The apparent affinity of the antibody assessed either by surface plasmon resonance or by functional titration on its agonist-like activity showed a similar value (10(8) M(-1)). The antibody recognized the receptor in its native form as shown by immunofluorescence experiments on A431 cells but not in its denatured form as shown by its absence of staining in immunoblots. The positive chronotropic effect in vitro was specifically blocked by both the antigenic peptide and the specific beta2-antagonist (+/-)-1-[2,3-(Dihydro-7-methyl1H-inden-4-yl)oxy]-3-[(1-methy lethyl)amino]-2-butanol hydrochloride (ICI1118,551). This activity was mediated through activation of Ca2+ L-type channels as assessed in guinea pig cardiomyocytes. These results suggest that the epitope is located in an extracellular alpha-helix, whose recognition by the antibody could stabilize the receptor in its 'active' conformation.

Ruthenium red as an effective blocker of calcium and sodium currents in guinea-pig isolated ventricular heart cells.

1. The effect of ruthenium red on calcium and sodium currents was studied in guinea-pig isolated ventricular heart cells with the whole cell patch-clamp technique. 2. Ruthenium red very efficiently blocked the L-type calcium current in a dose-dependent manner. A significant block was observed for concentrations as low as 0.3 microM. Analysis of the dose-response curve with the logistic equation indicated an EC50 of 0.8 microM, a maximum inhibition of 85% reached at 5 microM, and a coefficient of 2.37. 3. There was no shift in the voltage-dependence of the Ca current activation, nor in that of its steady-state inactivation determined with a 1 s prepulse. However, removal of Ca current inactivation at positive voltage was considerably reduced in the presence of concentrations of ruthenium red above 1 microM. A slowing of the time-course of inactivation of the Ca current was also observed. 4. At 10 microM, a concentration generally used to block the sarcoplasmic Ca release channels or the mitochondrial Ca uptake, ruthenium red blocked 26.7+/-4.3% (n=8) of the sodium current, and slowed its inactivation time-course. No effect was observed on the voltage-dependence of the current activation or inactivation. The peak sodium current was also decreased at a 10 times lower concentration by 7.6+/-2.7% (n=3). 5. Thus, at concentrations used to assess intracellular Ca movements, ruthenium red induced in heart cells a significant block of both Ca and Na channels.

In vivo and in vitro inhibition of the L-type calcium current in isolated guinea-pig cardiomyocytes by the immunosuppressive agent cyclosporin A.

Cyclosporin A (CsA), an immunosuppressive agent used to reduce rejection after organ transplantation, induces secondary effects in heart tissue. We have studied the effects in vivo and in vitro of CsA on L-type Ca2+ current (ICa) and the associated gating currents of isolated guinea-pig ventricular myocytes using the whole-cell patch-clamp technique. For in vivo experiments, a group of animals (n=28) was treated for 21 days by subcutaneous injection of CsA (15 mg/kg/day). Blood level of CsA was 1191+/-221 ng/ml (n=9). In cells from these animals (n=65, 19 animals), ICa was reduced to about 75% of that recorded from control cells (n=32, six animals). CsA decreased the availability of Ca2+ channels at potentials more positive than +30 mV. Isoproterenol (100 nM) was still able to increase ICa but only by 30+/-6% (n=9), whereas in control it increased ICa by 290+/-22% (n=5). Gating currents related to L-type Ca2+ channels were not altered in cells from CsA-treated animals. In in vitro experiments, CsA reduced ICa when applied directly to cardiomyocytes. CsA affected the kinetics of ICa inactivation, slowing down the rapid phase and accelerating the slow phase (n=4). The steady-state inactivation curve of ICa was shifted to more negative voltages and the degree of availability at -80 mV decreased by in vitro application of CsA. The half inactivation potential (V1/2) changed from -23+/-0.6 mV in control to -31+/-2 mV, -48+/-0.6 mV and -49+/-0.6 mV, in 1, 50 and 80 microM CsA, respectively. In these cells, the gating currents related to L-type Ca2+ channels were also not altered by CsA. CsA does not modify the Ca2+ channel density, although it induces a decrease in the beta1-adrenergic stimulation of ICa. The results are explained by a direct effect on the calcium channel inactivation of CsA and a non specific indirect effect.

Anti-peptide antibodies sensitive to the 'active' state of the beta2-adrenergic receptor.

Antibodies directed against a peptide corresponding to the second loop of the human beta2-adrenergic receptor were induced in rabbits by immunisation with the free peptide in complete Freund's adjuvant. The resulting antibodies were affinity-purified and shown to be monospecific for the target receptor. They were able to stimulate the L-type Ca2+ channels in whole-cell patch-clamp experiments on isolated adult guinea-pig cardiomyocytes. This effect was similar to that obtained by the specific beta2-adrenergic agonist zinterol. The antibody effects could be blocked with the specific beta2-adrenergic inverse agonist ICI118,551 but not with the neutral antagonist alprenolol. These results suggest that the antibodies recognise the active conformer of the beta2-adrenergic receptor.

Antibodies from Trypanosoma cruzi infected mice recognize the second extracellular loop of the beta 1-adrenergic and M2-muscarinic receptors and regulate calcium channels in isolated cardiomyocytes.

Sera from T. cruzi infected mice were tested in an enzyme immunoassay on peptides corresponding to the second extracellular loops of the beta 1-, the beta 2-adrenergic receptor and the M2 muscarinic receptor. All sera of mice (4/4) in the acute phase recognized the beta 1-adrenergic receptor and the M2 muscarinic receptor peptides but not the beta 2-adrenergic receptor peptide. The same peptides were recognized during the chronic phase in half of the mice (6/12). The immunoglobulin fractions of the mice were tested for their activity on L-type Ca++ channels of isolated guinea-pig cardiomyocytes using the whole-cell patch clamp technique. The immunoglobulin fractions of acute phase mice were able to activate the Ca++ channels by stimulation of the beta-adrenergic receptors, as assessed by inhibition with propranolol. Those of the chronic phase mice reduced the Ca++ current by stimulation of the muscarinic receptors, as assessed by inhibition with atropine. These results confirm the existence of functional epitopes on the second extracellular loops of both receptors. They suggest that, as in humans, the parasite is able to elicit functional autoantibodies against these epitopes. They give evidence that these autoantibodies mediate their physiological effects by modulating the cAMP activated Ca++ channels.

Effect of ryanodine on cardiac calcium current and calcium channel gating current.

The effects of 100 microM ryanodine on the L-type calcium channel were studied using the pacth-clamp technique in isolated guinea pig ventricular myocytes. The inactivation kinetics of the calcium current were slowed down in the presence of ryanodine in agreement with the blockade of the release of calcium from the sarcoplasmic reticulum by the drug. The I-V and steady-state inactivation curves of the calcium current were shifted to negative values by ryanodine. A similar shift was observed in the activation and inactivation curves of the intramembrane charge movement associated with the calcium channel. Due to this shift, ryanodine slightly reduced the maximal amount of displaced charge although it did not modify the transition from the inactivated to the activated state (i.e., charge movement repriming). This result is in notable contrast with that obtained in skeletal muscle, where it has been found that ryanodine interferes with charge movement repriming. These results provide additional evidence of the postulated differences between the architecture of the excitation-contraction coupling system in cardiac and skeletal muscle.

Effect of sulfhydryl oxidation on ionic and gating currents associated with L-type calcium channels in isolated guinea-pig ventricular myocytes.

OBJECTIVE: L-type calcium currents (ICa) and gating currents modification by extracellular application of the selective free sulfhydryl oxidant p-hydroxy-mercuric-phenylsulphonic acid (PHMPS) were studied. METHODS: Both currents were obtained with the whole cell patch clamp technique in guinea-pig ventricular cardiocytes. RESULTS: The main finding was a reduction of ICa clearly differentiable from a "run down" process. This effect was protected, stopped and in some cases partially reversed by dithiothreitol, a protective reagent for -SH groups. We also found a decrease of the gating currents associated with L-type calcium channels. The calcium modulation and cAMP phosphorylation systems of ICa are unaffected by PHMPS. With barium as charge carrier the current-voltage curves of barium currents were shifted by 10 mV to the positive direction by PHMPS. The same effect was obtained with calcium currents using BAPTA as a fast calcium buffer. CONCLUSION: The results indicate that oxidation of -SH groups carried by the channel protein induces dysfunction of the calcium entry to cardiac cells by altering the gating process. A participation of thiol functions on the gating of the calcium channel is proposed.

The stretch-activated ion channel blocker gadolinium also blocks L-type calcium channels in isolated ventricular myocytes of the guinea-pig.

We show that gadolinium (Gd3+) is a potent calcium channel blocker in guinea-pig isolated ventricular myocytes. A dose-dependent inhibition of ICaL was found with an EC50 of 1.4 microM and a complete inhibition at 10 microM Gd3+. When compared with Cd2+, it appeared that the blockade of ICaL is a complex phenomenon probably involving more than one site of interaction (a Hill coefficient of 1.6 was found for Gd3+ vs. 1.0 for Cd2+). It is concluded that Gd3+ ions completely block ICaL at concentrations used to block stretch-activated channels (SAC), rendering its use as a specific SAC inhibitor problematic.

The effects of increasing cell length on auxotonic contractions; membrane potential and intracellular calcium transients in single guinea-pig ventricular myocytes.

Until recently the investigation of length-dependent effects in cardiac muscle was restricted to multicellular preparations. We describe our experimental set-up which for the first time, in single cardiac myocytes, permits the effects of changes in cell length on auxotonic contractions (measured by carbon fibre transducers) to be simultaneously recorded with the effects on membrane potential and/or changes in intracellular calcium concentration (using indo-1 AM, acetoxylmethyl form). Consistent with previous findings (in experiments at 20-25 degrees C and 0.25 Hz) we report that following a stretch there was an increase in passive tension and contraction. A stretch which increased sarcomere length by approximately 3% had no significant effect on resting membrane potential or action potential amplitude. There was, however, a significant decrease in the action potential duration (P < 0.01, n = 8). No significant change in the amplitude of the intracellular calcium transient was seen following a stretch but a reduction in its duration was observed (P < 0.025, n = 11). Our observations on intracellular calcium transients are consistent with the hypothesis that, in mechanically loaded preparations, their time course is more dependent on changes in tension than changes in length.

A simple method for calibrating collagenase/pronase E ratio to optimize heart cell isolation.

A mixture of crude collagenase and non-specific proteases has been used to isolate guinea pig ventricular heart cells. Measurements of collagenase activity with Wünsch's substrate and protein content with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggest that collagenase enzymes do not play a major role in heart cell isolation. On the other hand, an important factor in heart digestion seems to consist of some fractions of the proteases present in crude collagenase. It is also noted that crude collagenases do not present any sensitivity to added calcium but because this ion is important to obtain isolated cells its role is discussed. According to our results, the SDS-PAGE method can be used to determine the appropriate enzyme concentrations to obtain calcium-tolerant myocytes. These myocytes have electrophysiological properties as reported in the literature.

Alteration of the L-type calcium current in guinea-pig single ventricular myocytes by heptaminol hydrochloride.

1. The effects of heptaminol on calcium current amplitude and characteristics were studied in single ventricular myocytes of guinea-pig by use of the whole cell configuration of the patch clamp technique. 2. A concentration-dependent decrease in ICa amplitude was observed. At heptaminol concentration as low as 10(-6) M, this effect was observed in only two cells (n = 6). At 10(-5) M the reduction of ICa was of 30 +/- 15% (n = 11). 3. The current recovery from inactivation at -40 mV holding potential (HP) seemed less sensitive to perfusion with heptaminol (greater than 10(-6) M). However, at -80 mV HP the overshoot of the recovery curve was decreased by heptaminol. 4. Both at -40 mV and -80 mV HP, heptaminol (10(-5) M) significantly increased the steady state inactivation of ICa. 5. As previously proposed by others to explain the effects of membrane active substances, the effects of heptaminol may result from alterations in cell membrane properties and possibly from an increase in intracellular free calcium ion concentration.

Rate dependence of action potential duration and calcium current in isolated guinea-pig cardiocytes.

The duration of the action potential at 50% of its amplitude (APD50) and peak calcium currents (ICa) was measured in single cardiac guinea-pig ventricular cells, using the whole-cell patch-clamp technique in current-clamp and voltage-clamp modes respectively. In the absence of intracellular calcium buffer, pacing at 0.28 Hz from a rest period of 1-2 min induced a transient increase (15.5 +/- 3.5%) in APD50, followed by a gradual shortening. Switching from 0.28 to 0.75 Hz again induced a transient increase of APD50 (6.8 +/- 2.9%). In the presence of EGTA or BAPTA on the cytosolic side of the membrane, this transient phase was prolonged and its amplitude slightly increased (10.6% when switching from 0.28 to 0.75 Hz in 5 mM-BAPA). The same increase in rate induced either a negative or a positive staircase of ICa, depending on the holding potential. At a holding potential of -80 mV, ICa peak was enhanced and the inactivation kinetics was slowed down. This facilitation of ICa seems to be dependent on calcium ions entering the cell via the calcium channels and could partly explain the observed transient increase in APD50.

Stretch-induced increase of resting intracellular calcium concentration in single guinea-pig ventricular myocytes.

Single guinea-pig ventricular myocytes were loaded with the fluorescent Ca2+ indicator Indo-1 AM and stretched by carbon fibres. Stretching increased resting tension. Sarcomere lengths were increased by 2-18%. It was observed that a stretch increased resting [Ca2+]i in seven out of eight cells. The change in [Ca2+]i increased with the size of the stretch and returned to pre-stretch levels on return to resting cell length. These observations suggest a means by which changes in resting muscle length can modify the contractile state of cardiac muscle.