Autocrine expression and ontogenetic functions of the PACAP ligand/receptor system during sympathetic development.

The superior cervical ganglion (SCG) is a well-characterized model of neural development, in which several regulatory signals have been identified. Vasoactive intestinal peptide (VIP) has been found to regulate diverse ontogenetic processes in sympathetics, though functional requirements for high peptide concentrations suggest that other ligands are involved. We now describe expression and functions of pituitary adenylate cyclase-activating polypeptide (PACAP) during SCG ontogeny, suggesting that the peptide plays critical roles in neurogenesis. PACAP and PACAP receptor (PAC(1)) mRNA's were detected at embryonic days 14.5 (E14.5) through E17.5 in vivo and virtually all precursors exhibited ligand and receptor, indicating that the system is expressed as neuroblasts proliferate. Exposure of cultured precursors to PACAP peptides, containing 27 or 38 residues, increased mitogenic activity 4-fold. Significantly, PACAP was 1000-fold more potent than VIP and a highly potent and selective antagonist entirely blocked effects of micromolar VIP, consistent with both peptides acting via PAC(1) receptors. Moreover, PACAP potently enhanced precursor survival more than 2-fold, suggesting that previously defined VIP effects were mediated via PAC(1) receptors and that PACAP is the more significant developmental signal. In addition to neurogenesis, PACAP promoted neuronal differentiation, increasing neurite outgrowth 4-fold and enhancing expression of neurotrophin receptors trkC and trkA. Since PACAP potently activated cAMP and PI pathways and increased intracellular Ca(2+), the peptide may interact with other developmental signals. PACAP stimulation of precursor mitosis, survival, and trk receptor expression suggests that the signaling system plays a critical autocrine role during sympathetic neurogenesis.

The relationship between temperature and calcium in acute cell damage after exposure to radiofrequency or thermal energy in isolated neonatal and adult rabbit cardiac myocytes.

Radiofrequency (RF) ablation is a nonsurgical technique using catheter-directed RF energy for treating cardiac arrhythmias in children and adults. Previous reports have suggested that sequestration of calcium (Ca2+) by the sarcoplasmic reticulum may partially protect mature cardiac myocytes from the effects of RF energy. The purposes of this study were to determine whether differences exist between neonatal and adult myocyte responses to RF energy and if myocyte damage is a Ca2+-dependent process. Because immature myocardium is functionally deficient in sarcoplasmic reticulum, we hypothesized that immature myocytes would be more susceptible to damage induced by RF energy. Isolated ventricular myocytes were obtained from neonatal and adult New Zealand White rabbits by enzymatic dissociation, then placed in a perfusion chamber designed to deliver RF energy or a heated perfusate solution. Measurements of bath temperature, cell morphology, and contractile response to electrical stimuli were recorded. RF energy application associated with increased perfusate temperature resulted in cell death, but not when the temperature rise was inhibited. Thus, the acute damage to cells exposed to RF energy appears to be mediated by thermal energy. After exposure to thermal energy, neonatal cells underwent contracture at lower temperatures than did adult cells. Perfusion with solutions containing low Ca2+ concentrations, comparable to intracellular diastolic Ca2+ levels, had a protective effect for both neonatal and adult myocytes. These findings indicate that acute cell damage after exposure to RF energy is mediated by a Ca2+-dependent process. Furthermore, immature myocardium is particularly susceptible to RF-mediated cell damage, possibly secondary to reduced Ca2+ sequestration by the sarcoplasmic reticulum.

Caffeine-induced contractions in developing rabbit heart.

Mature myocardium utilizes calcium released by the sarcoplasmic reticulum (SR) for cell contraction. Transient exposure of mature myocytes to caffeine is known to directly trigger Ca2+ release from the SR. In contrast, neonatal rabbit heart cells rely on transsarcolemmal Ca2+ influx for tension generation. SR function is decreased in immature heart and appears to play a minimal role as a calcium source. Accordingly, we hypothesized that neonatal rabbit myocytes would not respond to a caffeine pulse. Isolated neonatal and adult myocytes were paced to load the SR with calcium and then exposed to a 1-s pulse of 10 mM caffeine. As previously described, adult myocytes exhibited a brisk contraction in response to caffeine. Unexpectedly, neonatal myocytes also exhibited a similar, brisk response. These caffeine-induced contractions were not dependent on extracellular Ca2+ but were dependent upon the loading of SR Ca2+ stores. When SR Ca2+ stores were depleted by exposure to caffeine, mature myocytes exhibited only small, slow contractions in response to electrical field stimulation. Replenishing the SR Ca2+ stores resulted in normal, brisk contractions. In contrast, electrically stimulated contractions in immature myocytes were largely unaffected by caffeine-induced SR depletion. Thus, although neonatal myocytes are capable of loading and releasing calcium from the SR, such SR calcium release is not normally required for contraction in the developing heart. The minor role of SR Ca2+ release in immature rabbit heart may not result from immaturity of the SR, but rather from an inadequate mechanism to trigger SR calcium release.

Mechanisms of amiodarone-induced inhibition of Ca2+ current in isolated neonatal rabbit ventricular myocytes.

BACKGROUND: Amiodarone is an effective antiarrhythmic drug used to treat a wide variety of ventricular and supraventricular tachyarrhythmias. Recent voltage clamp studies indicate that amiodarone may possess a variety of antiarrhythmic effects. METHODS: The tight-seal, whole-cell voltage clamp technique was used to investigate the acute effects of amiodarone on L-type Ca2+ channel kinetics in isolated neonatal ventricular myocytes. RESULTS: We found that acute perfusion with 1 mumol/L amiodarone inhibited peak inward Ca2+ current by 39.1% (4.85 +/- 0.42 to 2.95 +/- 0.6 pA/pF, n = 10, p < 0.001) without changing the shape of the current-voltage relation. In addition, amiodarone shifted Ca2+ channel steady-state inactivation to more negative membrane potentials. In the absence of amiodarone, half inactivation of the Ca2+ current occurred at a membrane potential of -23.8 +/- 0.2 mV compared to -34.2 +/- 0.6 mV after addition of amiodarone (n = 11, p < 0.01). Furthermore, amiodarone significantly delayed Ca2+ current recovery from previous inactivation. CONCLUSIONS: These results provide evidence that amiodarone blocks voltage-dependent Ca2+ current in isolated neonatal rabbit ventricular myocytes by a variety of different mechanisms. The inhibitory effect of amiodarone on L-type Ca2+ current may represent an important facet of amiodarone's acute antiarrhythmic activity in the immature heart.

Developmental changes in the effects of pH on contraction and Ca2+ current in rabbit heart.

Protons inhibit Ca2+ current and contraction in heart muscle. The present study compares the effects of lowering the pH of the bath solution on single-cell contraction, action potential configuration and Ca2+ currents between neonatal and adult rabbit hearts. We found that a reduction of extracellular pH from 7.3 to 6.3 decreased cell contraction amplitude to 84.3% of control in isolated neonatal myocytes. A comparable change in extracellular pH resulted in a decrease in cell contraction to 56.2% of control in adult cells. Similarly, tension generation in intact neonatal papillary muscles was less sensitive to a decrease in external pH as compared to papillary muscles from adult animals. In addition, acidosis caused a less pronounced inhibition of Ca2+ current in neonatal cells than in adult cells (85 +/- 4% nu 62 +/- 4% of control, pH = 6.3, P < 0.001; 63 +/- 5% nu 32 +/- 5% of control, pH = 5.8, P < 0.001). Thus, the effect of external acidosis on myocardial contractility is commensurate with the effect on trans-sarcolemmal Ca2+ current. The membrane potential at which peak Ca2+ current occurred was not altered by low pH in neonatal cells but was shifted toward positive potentials by 17.7 mV in adult myocytes. Further, low external pH solution reduced Ca2+ current conductance more in adult cells than in neonatal cells. Moreover, action potential configuration in neonatal cells was altered less by acidosis as compared with adult cells. These findings may help explain the greater resistance of neonatal hearts to extracellular acidosis.

Adenylyl cyclase integrates multiple G protein signals to modulate calcium currents in neonatal rabbit heart.

We investigated the effects of added beta gamma subunits of G proteins (G beta gamma) on beta-adrenergic responsiveness of transmembrane Ca2+ currents (ICa) in ventricular myocytes from neonatal rabbits. G beta 1 gamma 1 purified from retinal rods was dialyzed into cells via the voltage clamp micro-electrode. Stimulation of ICa by isoproterenol was not affected by added intracellular G beta 1 gamma 1 or by carbachol alone but was completely blocked by combined G beta 1 gamma 1 and carbachol. Pretreatment of cells with pertussis toxin or temporal separation of carbachol and isoproterenol allowed stimulation of ICa by isoproterenol in cells dialyzed with G beta 1 gamma 1. Carbachol and G beta 1 gamma 1 together also did not prevent stimulation of ICa by dibutyryl-cyclic AMP. Thus, rather than simply inactivating Gs alpha by mass action, G beta 1 gamma 1 acts in concert with carbachol to inhibit isoproterenol stimulation of ICa.

ATP-sensitive potassium channels in neonatal and adult rabbit ventricular myocytes.

The properties of the ATP-sensitive potassium (KATP) current were studied in freshly isolated rabbit ventricular myocytes using the patch clamp technique. Removing ATP from the bath (intracellular) solution activated a large K+ conductance in patches from neonatal cells with properties similar to those of KATP channels in other preparations. In membrane patches from neonatal ventricular myocytes, the density of KATP channels was higher than the density of inwardly rectifying K+ channels and the mean patch KATP current was approximately 10 times that of the inwardly rectifying K+ current, at a patch membrane potential of -60 mV. Glibenclamide (10 microM) in the bath solution decreased the number of functional KATP channels, the open-state probability, and the mean patch membrane current. The single-channel conductance of the KATP channel was dependent on the external K+ concentration, and the relationship between channel conductance and external K+ concentration was fit by an exponential equation. In addition, the voltage dependence, channel density, and open-state probability of this channel were compared between neonatal and adult isolated ventricular myocytes. The single-channel conductance and channel density of the KATP channel in neonatal myocytes were significantly smaller than in adult cells. These results suggest that age-related changes occur in the properties of KATP channels.

Developmental changes in membrane Ca2+ and K+ currents in fetal, neonatal, and adult rabbit ventricular myocytes.

Whole-cell calcium current (ICa) and inwardly rectifying potassium current (IK1) were studied in 21-day fetal, 28-day fetal (total gestation, 31 days), 2-5-day neonatal, and adult rabbit ventricular myocytes isolated by enzymatic dissociation. Whole-cell peak ICa and IK1 at -100 mV increased significantly after birth. Cell size approximated from cell membrane capacitance also increased with age, with the most significant increase occurring after birth. When normalized to cell surface area, peak ICa density increased from day 21 of gestation to the neonate and then increased again from neonate to adult. In all age groups, peak ICa occurred at a test potential of +10 mV, and the shape of the Ca2+ current-voltage relation did not change with age. These findings suggest that there are no significant developmental changes in the voltage dependence of ICa. Therefore, the measured age-related increase in Ca2+ current density may result from increased channel expression. IK1 also exhibited a pattern of increasing current density with age. For IK1, the increase in current density was most rapid between day 21 and the perinatal period and much slower after birth. These results demonstrate that ICa and IK1 undergo significant changes during late fetal and postnatal development.

Calcium current measurements in acutely isolated neonatal cardiac myocytes.

Action potentials and voltage clamp-induced ionic currents were recorded in acutely isolated neonatal rabbit cardiac myocytes using the whole-cell voltage clamp technique. Time- and voltage-dependent Ca2+ currents in neonatal myocytes were elicited by depolarizations from a holding potential of -80 mV to various clamp potentials. The maximal measured inward Ca2+ current was 206 +/- 10 pA (mean +/- SEM, n = 51). The peak current occurred at a mean membrane potential of 7.8 +/- 1.3 mV (n = 51). The Ca2+ current voltage relation was shifted 26 mV in the positive direction when the external Ca2+ concentration was increased 10-fold. Ca2+ current rundown was observed with a half-time of approximately 20 min. Cells dialyzed with solution containing the Ca2+ chelating agent, EGTA (0.04 mM), had action potential durations similar to those previously reported in papillary muscle. In contrast, a higher concentration of EGTA (14 mM) prolonged the action potential duration. Control of the cell internal ionic composition was achieved by dialysis of the cell with a time constant for Na+ ions of 1.2 to 2.6 min. Tetrodotoxin (10 microM), included in some experiments to block Ca2+ entry via Na+ channels, was shown to be more than 98% effective. These results characterize the whole-cell voltage clamp technique as applied to immature heart cells.

Calcium current and tension generation in immature mammalian myocardium: effects of diltiazem.

Single sucrose gap and isolated myocyte voltage-clamp techniques were used to study the effects of diltiazem on calcium current (ICa) and tension generation in isolated ventricular myocytes and right ventricular papillary muscles from neonatal New Zealand White rabbits. Diltiazem was shown to significantly shorten the duration of isolated myocyte action potentials with no effect on overshoot potential or resting membrane potential. Diltiazem blocked but did not completely abolish ICa in these neonatal cells. Addition of diltiazem to the solution bathing papillary muscles resulted in a similar reduction in action potential duration accompanied by a reduction in twitch tension. When the duration of depolarization was controlled employing the single sucrose gap voltage clamp, the decrease in tension development caused by diltiazem was abolished despite a significant decrease in twitch tension in the same muscles. In another series of experiments it was demonstrated that the magnitude of developed tension in neonatal papillary muscles is dependent upon the duration of depolarization. Taken together, the results of this investigation suggest that in neonatal myocardium when ICa is blocked by diltiazem, the resulting reduction in developed tension is caused in part by reduction of action potential duration. The calcium carried into the neonatal heart cell by ICa does not appear to be the only source of extracellular Ca2+ for excitation-contraction coupling. Finally, the action potential appears to act as a gate for calcium movement into the neonatal heart cell.

Single-channel recording of inwardly rectifying potassium currents in developing myocardium.

Properties of the inwardly rectifying K+ channel, which contributes to the maintenance of the resting membrane potential, were studied in neonatal rabbit ventricular myocytes using the patch-clamp technique. Inward rectification was evident in single-channel current-voltage (I-V) relations at potentials positive to the potassium equilibrium potential (Ek = 0 mV with [K+]o = [K+]i = 150 mM, [Mg2+]i = 2 mM). The single-channel conductance was 3.2 +/- 0.1 pS in physiological (5.4 mM) [K+]o. The zero-current potential shifted 48.4 +/- 2.4 mV for a ten-fold change in [K+]o in neonatal cells. External Ba2+ blocked the current in a dose-dependent manner. The voltage dependence, open-state probability and channel density of this channel were compared between neonatal and adult ventricular myocytes isolated by similar techniques. The open-state probability of the channel was approximately the same in neonatal (0.39 +/- 0.06, n = 13) as in adult cells (0.4 +/- 0.05, n = 11). However, in symmetrical transmembrane K+ concentration [( K+]o = [K+]i = 150 mM), the single channel conductance was significantly smaller in neonatal (25 +/- 0.3 pS, n = 25) as compared with adult cells (31 +/- 0.4 pS, n = 12). In addition, the relationship between resting membrane potential and [K+]o was measured in neonatal and adult myocytes. The resting membrane potential in the neonate was less dependent on [K+]o than in the adult. These results are consistent with an age-related change in resting membrane K+ permeability which may result from a developmental change in the single-channel conductance properties of the inwardly rectifying K+ channel.

Developmental changes in cardiac myocyte calcium regulation.

Developmental changes in the contributions of transsarcolemmal Ca2+ influx and Ca2+ release from intracellular storage sites to myocardial contraction were evaluated in isolated ventricular myocytes from neonatal (aged 1-7 days) and adult (aged 8-10 weeks) New Zealand White rabbits. Contractions ceased in one beat when extracellular Ca2+ was decreased from 1mM to micromolar levels using a rapid perfusion technique. On reperfusion with 1 mM Ca2+, recovery of control contraction amplitude occurred after significantly fewer beats in neonatal myocytes compared with adult myocytes, and after 1 minute compared with 5 minutes of reduced Ca2+. After 15 minutes of perfusion with either 1 or 10 microM ryanodine, contraction amplitude decreased in both age groups, but the decrease was significantly greater in adults than in neonates. These experiments indicate that isolated ventricular myocytes may be used in the study of developmental changes in intracellular Ca2+ regulation. Results suggest that cardiac contraction in neonates is relatively more dependent on transsarcolemmal Ca2+ influx. Furthermore, although Ca2+ release from intracellular storage sites is present in both neonates and adults, its role in cardiac contraction is more significant in adults.

Ethics in cardiovascular medicine. Task Force IV: Scientific responsibility and integrity in medical research.

Ethical standards are a set of affirmative responsibilities to which the investigator must subscribe; behavior that is incompatible with these responsibilities should be presumed unethical, whether or not it is explicitly proscribed. This Task Force sought to present these standards as principles or guidelines. In undertaking research an investigator must accept that publicly funded or supported research is intended to yield public benefit; personal gain should be only incidental to and not at the expense of the public benefit. The responsibilities of the investigator are summarized as follows: Design of Research To develop a research design that effectively and efficiently addresses the scientific question while minimizing the likelihood of incorrect or misleading results. To protect the rights and welfare of human subjects, assure the humane use of laboratory animals and protect the safety of laboratory workers and the environment. Conduct of Research To ensure that accepted laboratory and research practices are followed and that all data are accurately collected and properly recorded; the investigator must participate in the review of original data. To carry out research in accordance with that approved by the institutional review board and ensure that fully informed consent is obtained, that the welfare of human subjects is protected and that animal welfare and laboratory safety procedures are carried out. To provide effective ongoing supervision of research trainees and technicians. In multidisciplinary collaborative research, to have at least an overview familiarity with the work outside his or her areas of expertise. In fixed protocol, multicenter collaborative research the investigator must be satisfied with the adequacy of the collaborative activities.(ABSTRACT TRUNCATED AT 250 WORDS)

The biphasic effect of amrinone on tension development in newborn mammalian myocardium.

The effect of the bipyridine compound, amrinone, on tension generation in neonatal and adult myocardium was studied over the concentration range 30-500 micrograms/mL. Increasing concentrations of amrinone caused a monotonic increase in twitch tension and the rate of tension development in adult papillary muscles. In contrast, lower concentrations of amrinone (30 and 100 micrograms/mL) caused a decrease in twitch tension and dP/dt in newborn papillary muscles, whereas 500 micrograms/mL amrinone caused a significant increase in both parameters in the younger age group. Lactic acid, used to dissolve amrinone, was shown to have no effect on tension development. Half relaxation time was decreased in adult preparations at all concentrations of amrinone. In comparison, the decrease in half relaxation time produced by amrinone in the newborn was significant only at a concentration of 500 micrograms/mL. Action potential duration in the newborn was significantly shortened by 30 micrograms/mL amrinone. In voltage clamp experiments, 30 micrograms/mL amrinone was shown to have no effect on tension accompanying two second voltage clamp steps to the plateau potential in newborn myocardium. Developed tension at 400 ms into the clamp step, final tension, and the ratio of early peak tension to final tension were all unchanged by the low concentration of amrinone. In contrast, 500 micrograms/mL amrinone in the newborn increased tension at 400 ms and final tension but had no effect on the ratio of early peak tension to final tension. These results suggest that the negative inotropic effect of lower concentrations of amrinone on neonatal myocardium is the result of changes in action potential configuration and not a true alteration in basic mechanisms of intracellular Ca2+ regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

A diminished role for the sarcoplasmic reticulum in newborn myocardial contraction: effects of ryanodine.

Ryanodine, known to reduce Ca2+ release from the sarcoplasmic reticulum (SR), was used in conjunction with the single sucrose gap voltage clamp technique to study excitation-contraction coupling in papillary muscles isolated from newborn and adult rabbits. Ryanodine altered the components of voltage clamp-induced tension in the adult producing a pattern that closely resembled the normal newborn. The phasic component of tension in mature myocardium was markedly and significantly reduced by ryanodine. In addition, ryanodine significantly altered the voltage dependence of this component of tension, suggesting a change in its mechanism of generation. The appearance of a prominent phasic component of tension, absent normally, but produced in the newborn by Ca2+ loading, was also abolished by ryanodine. These results support previous proposals that the phasic component of voltage clamp-induced tension in mammalian myocardium is produced by the release of Ca2+ from the SR. In the newborn, ryanodine caused a significantly smaller decrease in the ratio of phasic to tonic tension than in the adult, suggesting a much less significant role for the SR in excitation-contraction coupling in the younger age group. Although ryanodine had a significant effect on phasic tension, no alteration in the amplitude or voltage dependence of tonic tension, generated by transmembrane Ca2+ influx, could be demonstrated in either newborn or adult heart. This investigation suggests that in newborn myocardium, Ca2+ release from the SR plays a negligible role in excitation-contraction coupling, which depends, rather, on the influx of Ca2+ from the extracellular space.

Bilateral trochlear nerve paresis in hydrocephalus.

Three patients with nonneoplastic hydrocephalus had bilateral paresis of the trochlear nerves. Associated signs, including paresis of upgaze, light-near dissociation of the pupils, and convergence-retraction nystagmus, suggested rostral involvement of the mesencephalon. Trochlear nerve paresis and accompanying signs improved after revision of ventricular shunts in two patients. Bilateral trochlear nerve paresis may be a localizing sign of involvement of the superior medullary velum (the anatomic site of trochlear nerve decussation) by a dilated sylvian aqueduct and/or downward pressure from an enlarged III ventricle.

Cardiac arrhythmias: the role of pharmacologic intervention.

The growing menu of drugs used to treat arrhythmias in children enhances the importance of antiarrhythmic selection based on the application of underlying electrophysiologic and pharmacokinetic principles, as well as a reduction of the side effect to benefit ratio. No attempt has been made in this report to discuss the diagnosis of arrhythmias in children, nor is the list of agents we discuss all-encompassing. Rather, the major thrust has been to promote an understanding of the important relationship between anatomic considerations, basic electrophysiology, and developmental pharmacology in directing therapy. We wish to emphasize that many of the studies quoted in this report are investigational, and that not all of the applications listed in the text and tables are approved for use in pediatric patients in the United States. Nevertheless, the information may serve as a guideline to developing management strategies for the individual child with an arrhythmia. Given the number of new antiarrhythmic agents under development, it would be no surprise if, in a few years, a paper such as this one discussed several new drugs we have not mentioned. We hope that the principles we have outlined will serve as a framework for incorporating these new agents into clinical practice as they become available.

Excitation-contraction coupling in developing mammalian myocardium: evidence from voltage clamp studies.

The single sucrose gap voltage clamp technique was used to study excitation-contraction coupling processes in right ventricular papillary muscles from New Zealand White rabbits at various stages of development. In response to voltage clamp controlled depolarizations, muscles from newborn rabbits were found to exhibit a monotonically increasing tension response reaching a steady state level that was maintained for the duration of depolarization. In contrast, more mature myocardium responded to similar depolarizations by developing an early peak of tension before relaxing to a steady state level. Measurement of the ratio of early peak or phasic tension to steady state or tonic tension revealed a statistically significant increase in the phasic tension component with maturation. In addition, Ca2+ loading of immature myocytes via a conditioning voltage clamp step resulted in enhancement of phasic tension in subsequent test depolarizations. Finally, the voltage dependence of tonic tension was found to be the same in all age groups. In contrast, the voltage dependence of phasic tension, seen only in the more mature myocardium, differed from that of tonic tension. The results of this investigation suggest that tension development in the immature myocardium is supported largely by the influx of Ca2+ across the sarcolemma. As the myocardium matures, intracellular Ca2+ uptake and rerelease by the sarcoplasmic reticulum plays an increasingly important role in tension development. A developmental schema is presented to account for the observed maturational changes in excitation-contraction coupling.