Are developing beta-adrenoceptors able to desensitize? Acute and chronic effects of beta-agonists in neonatal heart and liver.

During fetal and neonatal development, beta-adrenergic receptors (beta-ARs) appear to be resistant to desensitization by beta-agonist drugs. To determine the mechanisms underlying the regulatory differences between adults and neonates, we administered isoproterenol, a mixed beta(1)/beta(2)-AR agonist, and terbutaline, a beta(2)-selective agonist. Effects were examined in the ensuing 4 h after a single injection, or after the last of four daily injections. We prepared cell membranes from heart (predominantly beta(1)-ARs) and liver (predominantly beta(2)-ARs) and assessed signal transduction in the adenylyl cyclase (AC) pathway. In the first few hours after a single administration of isoproterenol to adult rats, cardiac beta-ARs showed activation of G proteins (elevated AC response to forskolin) and desensitization of beta-AR-mediated responses; after the fourth injection, heterologous desensitization emerged, characterized by a loss of signaling mediated either through beta-ARs or glucagon receptors. Terbutaline evoked an increase in the forskolin response but no desensitization of receptor-mediated responses. When we gave the same treatments to neonatal rats, we observed cardiac G protein activation, but there was neither homologous nor heterologous desensitization of beta-ARs or glucagon receptors. In the adult liver, isoproterenol and terbutaline both failed to evoke desensitization, regardless of whether the drugs were given once or for 4 days. In neonates, however, acute or chronic treatment elicited homologous desensitization of beta-AR-mediated AC signaling, while sensitizing the response to glucagon. These results show that neonatal beta-ARs are inherently capable of desensitization in some, but not all, cell types; cellular responses can be maintained through heterologous sensitization of signaling proteins downstream from the receptor. Differences from adult patterns of response are highly tissue selective and are likely to depend on ontogenetic differences in subtypes of beta-ARs and AC.

Functional alterations in CNS catecholamine systems in adolescence and adulthood after neonatal chlorpyrifos exposure.

Chlorpyrifos (CPF), one of the most widely used pesticides, is a neurobehavioral teratogen in animals. We administered CPF to neonatal rats on postnatal days (PN) 1-4 (1 mg/kg) or PN11-14 (5 mg/kg), regimens devoid of overt systemic toxicity. We then examined the impact on catecholaminergic systems in adolescence (PN30) and adulthood (PN60), assessing basal neurotransmitter content and transmitter utilization rates (turnover) in brain regions comprising the major noradrenergic and dopaminergic projections. Although CPF had only sporadic effects on basal norepinephrine and dopamine content, it profoundly suppressed norepinephrine turnover across multiple regions, indicative of net reductions in presynaptic activity. Dopamine turnover showed less consistent effects, with subnormal turnover in some regions and activation in others. We also evaluated whether CPF exposure altered the ability of catecholamine systems to respond to acute cholinergic stimulation, elicited by administration of a single challenge dose of nicotine. In the normal brain, nicotine increases the utilization of norepinephrine and dopamine. With only a few exceptions, animals receiving neonatal CPF exposure showed lasting desensitization of the nicotine response; not only was the activation by nicotine blunted in the CPF group, but in some regions the nicotine response was reversed, eliciting a reduction in transmitter turnover. These results indicate that neonatal CPF exposure produces widespread deficiencies in catecholaminergic synaptic function that persist into adulthood, and that are best revealed by dynamic measures of synaptic activity and responsiveness, as opposed to static markers like basal transmitter levels. The effects seen here are likely to contribute to alterations in behavioral performance that persist or emerge long after the termination of CPF exposure.

Beta-adrenoceptor-mediated cell signaling in the neonatal heart and liver: responses to terbutaline.

Terbutaline, a beta(2)-adrenoceptor (beta(2)-AR) agonist, is a widely used tocolytic that also crosses the placenta to stimulate fetal beta-ARs. The current study examines the effects of terbutaline administered to neonatal rats. Terbutaline (10 mg/kg sc) given on postnatal day (PN) 2-5 or PN 11-14 elicited significant downregulation of both cardiac and hepatic beta-ARs, with a much greater effect in the liver. Despite the reduction in cardiac beta-ARs, receptor desensitization was absent as evidenced by the maintained ability of isoproterenol to stimulate adenylyl cyclase (AC) in membrane preparations. The underlying mechanism was dissected by using stimulants that operate at different points in the AC signaling pathway, NaF, forskolin, and Mn(2+). When administered in the early neonatal period, terbutaline failed to evoke any changes in cardiac AC activity; however, treatment on PN 11-14 evoked heterologous sensitization downstream from the receptor, evidenced by increases in the response to NaF and forskolin. In the liver, neonatal terbutaline administration elicited a small (approximately equal to 10%) decrease in the AC response to isoproterenol, an effect much smaller than the downregulation of beta-ARs (>40%). In this tissue, desensitization was again offset by heterologous sensitization of AC signaling. These results indicate that, in the developing organism, beta-AR-mediated cell signaling responses are maintained in the face of receptor downregulation through heterologous induction of downstream signaling elements. These unique responses serve to sustain beta-AR signaling in the perinatal period.

Beta-adrenoceptor signaling in the developing brain: sensitization or desensitization in response to terbutaline.

Beta(2)-adrenoceptor agonists are commonly used to arrest preterm labor but they also penetrate the placenta to stimulate fetal beta-adrenergic receptors (betaAR), and have been implicated in subsequent neurobehavioral deficits. We administered terbutaline to pregnant rats on gestational days (GD) 17-20 and during two postnatal (PN) periods, PN2-5 and PN11-14, that correspond to third trimester human neurological development. We then examined betaAR binding sites and adenylyl cyclase (AC) signaling in fetal brain or neonatal brain regions. Although fetal terbutaline administration evoked betaAR downregulation, the ability of isoproterenol to stimulate AC was enhanced instead of desensitized. Sensitization occurred at post-receptor signaling proteins, as augmented responses were also seen for stimulants that bypass the receptors to work on G-proteins (NaF) or that stimulate AC directly (forskolin and Mn(2+)). When terbutaline was given on PN2-5, betaAR downregulation was obtained in brainstem, forebrain and cerebellum, but desensitization of the AC response was seen only in the forebrain; the desensitization was heterologous, reflecting decrements in total AC activity rather than specific loss of the betaAR response. With treatment on PN11-14, only the cerebellum showed betaAR downregulation and induction at the level of post-receptor signaling proteins maintained the betaAR-mediated AC response. Our results indicate that, unlike the adult, betaAR signaling in the fetus and neonate is resistant to homologous desensitization by beta-agonists, and in fact, displays heterologous sensitization that sustains or enhances the overall response. The inability to desensitize betaAR responses may lead to disruption of neural cell development as a consequence of tocolytic therapy.

Persistent cholinergic presynaptic deficits after neonatal chlorpyrifos exposure.

The commonly-used organophosphate insecticide, chlorpyrifos (CPF), impairs brain cell development, axonogenesis and synaptogenesis. In the current study, we administered CPF to neonatal rats on postnatal (PN) days 1-4 (1 mg/kg) or PN11-14 (5 mg/kg), treatments that were devoid of overt toxicity. We then examined two cholinergic synaptic markers, choline acetyltransferase activity (ChAT) and [3H]hemicholinium-3 binding (HC-3) in the hippocampus, midbrain, striatum, brainstem and cerebral cortex in the juvenile (PN30) and young adult (PN60). Across all brain regions, CPF exposure evoked significant reductions in both markers, with larger effects on HC-3 binding, which is responsive to neuronal impulse activity, than on ChAT, a constitutive marker. Superimposed on the deficits, there were gender-selective effects and distinct regional disparities in the critical exposure period for vulnerability. In the hippocampus, either the early or late treatment regimen evoked decreases in ChAT but the early regimen elicited a much larger decrease in HC-3; effects persisted into adulthood. In the midbrain, CPF administration on PN1-4 elicited deficits similar to those seen in the hippocampus; however, exposure on PN11-14 elicited changes preferentially in females. Gender selectivity was also apparent in the striatum, in this case reflecting deficits in females after CPF treatment on PN1-4. In contrast, the effects of CPF on the brainstem were relatively more robust in males; effects in the cerebral cortex were less notable than in other regions. These results indicate that neonatal CPF exposure produces widespread deficiencies in cholinergic synaptic function that persist into adulthood. The effects are likely to contribute to gender-selective alterations in behavioral performance that persist or emerge long after the termination of exposure and well after the restoration of cholinesterase activity.

Gender and fat metabolism during exercise: a review.

We examined the evidence for greater fat utilization by women during exercise and the potential gender differences in specific cellular processes. Results from well-controlled studies show that, compared to men, women oxidize more fat during submaximal exercise, resulting in the relative sparing of muscle glycogen. Mature female rats use less muscle glycogen during running and can run longer than male counterparts. Circulating estrogen is critical to these observations, as shown by studies where male rats were treated with estrogen. Estrogen-treated male rats use less muscle glycogen during exercise and can run longer than untreated males. The cellular mechanisms and factors underlying these findings are unknown and certainly multifactorial. We offer some information that, unfortunately, does not lead to any natural conclusion. However, this area is certainly ripe for research.

Exercise training improves lusitropy by isoproterenol in papillary muscles from aged rats.

Aging is associated with a decreased cardiac responsiveness to beta-adrenergic stimulation. We examined the effect of endurance exercise training of old Fischer 344 male rates on beta-adrenergic stimulation of the function of isolated left ventricular papillary muscle. Three groups were examined: sedentary mature (SM; 12-mo old), sedentary old (SO; 23-24 mo old), and exercised old (EO; 23-24 mo old) that were treadmill trained for 4-8 wk. The isometric contractile properties were studied at 0.2 Hz and 0.75 mM calcium. Without beta-adrenergic stimulation, there were no group differences for peak tension, maximum rate of tension development (+dP/dt), or maximum rate of tension dissipation (-dP/dt). The time to peak tension was longer (P < 0.05) for both EO and SO than for SM rats. Half relaxation time (RT1/2) was prolonged (P < 0.05) for SO compared with SM and EO (which did not differ). The three groups did not differ in the beta-adrenergic stimulation by isoproterenol of peak tension, -dP/dt, time to peak tension, or contraction duration. The inotropic response (+dP/dt) of SM was greater (P < 0.05) than that in SO or EO rats (which did not differ); however, the lusitropic response (RT1/2) was lesser (P < 0.05) in SO than in SM or EO rats (which did not differ). Thus exercise training of old rats improved the lusitropic response to isoproterenol without altering the age-associated impairment in inotropic response.

SERCA2a and mitochondrial cytochrome oxidase expression are increased in hearts of exercise-trained old rats.

Aging of rats results in slower activities of calcium transport by cardiac calcium adenosinetriphosphatase (ATPase) of the sarcoplasmic reticulum (SR) and mitochondrial cytochrome oxidase (COX). These enzyme activities are faster after exercise training of previously sedentary old rats. Our purpose was to determine whether the expression of the genes encoding SR calcium ATPase (SERCA2a) or COX is altered by exercise training. Old (24-mo-old) male Fischer 344 rats were assigned to SO (sedentary old) or EO (exercised old) groups and compared with younger (12-mo-old) sedentary rats (SM). EO rats were trained on a treadmill for 8-10 wk. SERCA2a and COX mRNAs were lower (P < 0.05) in SO compared with SM and EO, whereas glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cardiac alpha-actin mRNAs were similar across groups. The immunoreactive protein contents of cardiac calcium ATPase, cytochrome c, sarcomeric actin, and GAPDH followed the changes, when observed, in mRNA contents. Thus pretranslational mechanisms may be modified in some genes during aging and exercise training of previously sedentary old rats.

Gender differences in the behavioral manifestations of Alzheimer's disease.

OBJECTIVE: To examine the relationship between gender and specific types of behavior problems that occur in patients with Alzheimer's disease. DESIGN: This was an observational study using the Dementia Behavior Disturbance Scale to quantify and define behavioral problems encountered by caregivers. Multiple regressions were used to control for the possible influence of dementia severity as measured by the Mini-Mental State Examination and the duration of dementia. SETTING: Patients were sampled from the outpatient dementia clinics of Roger Williams Hospital and Miriam Hospital in Providence, Rhode Island. PARTICIPANTS: A total of 125 patients with probable Alzheimer's disease, defined by NINCDS-ADRDA diagnostic criteria, were included in the study. There were 75 women and 50 men. MAIN OUTCOME MEASURES: Caregivers rated the presence and frequency of 28 different behavior problems from the Dementia Behavior Disturbance Scale. Domains of behavior disturbance were then defined by a factor analysis of the data. RESULTS: Male and female groups were comparable for the demographic variables of age, education, and duration of dementia, as well as severity of depression, degree of cognitive impairment and overall severity of behavior disturbance. Among the six behavior factors that were defined, two were significantly related to gender. One factor, which included apathy and vegetative signs, was related to male gender; a second factor, which included reclusiveness and emotional lability, was related to female gender. CONCLUSIONS: Although overall severity of behavior disturbance in Alzheimer's disease may be related primarily to severity in dementia, significant differences in the types of behaviors manifested exist between males and females with the disease.

A chronically instrumented rat model to assess the altered baroreflex due to exercise.

We developed a conscious, chronically instrumented, exercise-trained rat model and examined the time course of the training-induced alteration of baroreflex function in response to hypertensive conditions. Exercise-trained (ET) animals ran at 18 m.min-1, 15% grade, for 60 min.d-1, 5 d.wk-1 for 5 wk. Baroreflex tests were conducted on day 6 each week. Regression line slopes relating the change in mean arterial pressure (delta MAP) to the change in heart rate (delta HR) were used to assess baroreflex sensitivity. Intravenous injections of phenylephrine were used to create hypertensive conditions. Compared with the C group, slopes of ET animals were reduced (from 2.1 to 1.2 bpm.mm Hg-1, P < 0.05) as early as week 3 of training in response to increasing doses of PE, and reached 0.8 bpm.mm Hg-1 by the end of training. The reflex bradycardiac response (delta HR) to PE was reduced (P < 0.05) depending on the dose of PE and the duration of training: in micrograms PE.kg-1 body weight, 5 (71% +/- 6% of control at week 2), 3 (70% +/- 7% of control at week 3, and 1 (61% +/- 10% of control at week 4). The pressor (delta MAP) to PE remained constant throughout training. Thus, using a chronically instrumented rat model that maintains the ability to run, we observed that the ability of the arterial baroreflex to produce bradycardia during pressor events was substantially reduced following as few as 2 wk of training.

Age-related levels of GABA/benzodiazepine binding sites in cerebrum of F-344 rats: effects of exercise.

We examined gamma-aminobutyric acid (GABA), benzodiazepine and convulsant sites of postsynaptic GABA/benzodiazepine receptors (GBZR) in cerebral membranes of inbred Fischer 344 male rats as a function of age. In aged rats (23 to 24 months), the benzodiazepine binding site as determined by [3H]flunitrazepam was 47% and 43% lower than corresponding values in young adult (3 to 4 months) and mature (10 to 12 months) rats, respectively. The decrease was due to the loss of binding density rather than a change in affinity. No statistically significant age-related changes in [3H]muscimol binding were observed when 5 nM or 40 nM labeled muscimol were used. GABA produced a dose-dependent stimulation of flunitrazepam binding in all age groups, but the maximum stimulation in aged animals was significantly higher (24%) than in young and mature animals. The [35S]TBPS binding site, the convulsant site of GABA/benzodiazepine receptors, was unaffected with age. We also studied the effects of exercise on GBZR binding sites of aged rats. The decline of flunitrazepam binding sites and the high sensitivity of flunitrazepam binding to regulation by GABA in aged animals were reversed by 8 to 10 weeks of endurance exercise. Endurance exercise did not have any significant effect on muscimol or TBPS binding sites. Results suggest that there are aged-related alterations of GBZR binding sites and that these modifications can be reversed by exercise.

Mechanisms for the responses of cardiac muscle to physical activity in old age.

The decline of maximal cardiac output (Qmax) is a major factor responsible for the lower maximal oxygen consumption of elderly mammals. The lower Qmax is associated with aging-related decreases in maximal heart rate (HR-max) and maximal stroke volume (SVmax). The mechanism(s) for the slower HRmax, unchanged by exercise training, is unknown. The decrement in SVmax, however, can be improved, as shown by the enhanced systolic and diastolic properties of the elderly heart after exercise training. One major problem is diastolic dysfunction observed in the absence of disease. Diastolic dysfunction (a decrease in peak ventricular filling after systole or a prolonged relaxation of contracted muscle) results from in part a downregulation of the sarcoplasmic reticulum's (SR) calcium ATPase that sequesters cytosolic calcium via the hydrolysis of ATP. Exercise training of sedentary old mammals produces a faster relaxation and an upregulation of the SR calcium ATPase. Yet the characteristic shift of myosin toward the slower isoform is unaltered by exercise training. The molecular signals and mechanisms underlying these aging-related alterations in sedentary and physically active individuals are unknown. An enhancement of cardiac function by exercise training, though, is preserved in advanced age.

Mitochondrial metabolism and substrate competition in the aging Fischer rat heart.

OBJECTIVE: The objective was to examine mitochondrial oxidative metabolism of long chain fatty acids and to compare it with glucose uptake and the generation of pressure-volume work in hearts from mature and aged rats. METHODS: Hearts from mature (8 to 15 months of age) and old (28 to 30 months) Fischer 344 rats were perfused as working hearts with either 10 mM glucose or glucose plus 1 mM oleic acid (2% bovine serum albumin) and rates of glucose extraction were determined. Hearts were subjected to a stepwise increase in work load. In separate experiments, mitochondria were isolated from mature and old rat hearts and assayed for respiratory function, carnitine exchange, carnitine palmitoyltransferase activities, and phospholipid content. RESULTS: Although there were no differences in peak work attained between the mature and old rats in the presence of either glucose alone or glucose plus oleic acid, glucose utilisation was significantly decreased by oleate in the mature animals only. No significant changes in either glutamate or succinate (+rotenone) supported respiration were found in heart mitochondria isolated from old rats compared with mature animals. In agreement with prior studies with the Wistar rat model of aging, significant decrements in the rates of palmitoylcarnitine oxidation and carnitine exchange were apparent in the old Fischer animals. A significant lowering of heart mitochondrial carnitine palmitoyltransferase I activity was also found in the old animals. A decrease in the amounts of carnitine loaded in mitochondria from old animals is consistent with reduced carnitine content in both mitochondria and whole hearts from aged Wistar and Fischer rats. A significant (23%) reduction in heart mitochondrial cardiolipin content from 30 month old Fischer rats suggests that this phospholipid may also contribute to the lower rates of carnitine and acylcarnitine transport across the mitochondrial inner membrane. CONCLUSION: The limitation in the delivery of fatty acyl units to beta oxidation as measured in isolated heart mitochondria from old rats has a physiological correlate in the intact heart. The well documented suppression of glucose oxidation by fatty acids seen in the adult rat heart is not seen in old hearts, supporting the in vitro finding of decreased oxidation of palmitoylcarnitine with senescence.

CaATPase content is lower in cardiac sarcoplasmic reticulum isolated from old rats.

The rate of oxalate-facilitated ATP-dependent calcium uptake by the calcium pump, calcium adenosinetriphosphatase (CaATPase), is 30-40% slower in the sarcoplasmic reticulum (SR) isolated from the hearts of senescent Fischer 344 male rats. To determine the underlying mechanism, cardiac SR was isolated from 11- to 12-mo-old (adult) and 22- to 24-mo-old (senescent) male Fischer 344 rats. The yield of SR and contamination by other membrane organelles were similar between the groups. The rate of calcium uptake by the homogenate and isolated SR was 28-44% slower (P < 0.05) in the senescent group. In the isolated SR the calculated maximal velocity (Vmax) of CaATPase activity as a function of varying concentrations of ATP or calcium was 20-30% lower (P < 0.05) in the senescent group; however, the affinities for both calcium and ATP of CaATPase activity were unaltered. The lower Vmax was matched by a decreased (P < 0.05) content of calcium-dependent phosphoenzyme (EP) in the SR isolated from the senescent rats. Thus the ratio of enzyme activity to phosphoenzyme content (Vmax/EP) was similar between the groups. The immunoreactive CaATPase protein was 22 +/- 2% lower in the SR from the senescent rats. Taken together the data indicate that the major mechanism underlying the slower calcium transport by cardiac SR isolated from old rats is a lower content of the CaATPase protein.

The MgATPase activity of rat cardiac sarcoplasmic reticulum is a function of the calcium ATPase protein.

Magnesium-dependent ATPase (MgATPase) activity is associated with many E1-E2 or P-type transport ATPases including the sarcoplasmic reticulum (SR) calcium ATPase. The SR isolated from rat heart has a MgATPase activity which is 6-12 times faster than the MgATPase activity of the SR isolated from dog heart. To determine the origin of the high MgATPase activity of rat heart SR, we compared and contrasted cardiac SR isolated from both species. The preparations were similar in the following ways: (i) contamination by other organelles; (ii) the comigration of MgATPase activity with calcium-dependent ATPase (CaATPase) activity through a sucrose gradient; (iii) a similar ATPase activity sensitivity to pH and ATP concentration; (iv) the high and similar of sensitivity of ATPase activity to detergent; and (v) a similar protein profile. In both preparations, a single protein in the 105,000-Da region of polyacrylamide gels was phosphorylated by ATP, and the phosphorylated species was an acylphosphate formed in the presence and absence of calcium. Dimethyl sulfoxide, which slows acylphosphoenzyme breakdown, markedly inhibited both CaATPase and MgATPase activities of both preparations but not other enzyme activities. Importantly, the specific inhibitor of the SR calcium pump, thapsigargin, completely inhibited the CaATPase activity with an I50 of 6-7 nM; however, a higher concentration (I50 of 2 microM) was required to inhibit the MgATPase activity of the rat cardiac SR. These results provide evidence that the MgATPase activity of rat cardiac SR is part of the enzyme cycle of the calcium ATPase protein.

The role of dicarboxylic anion transport in the slower Ca2+ uptake in fetal cardiac sarcoplasmic reticulum.

Sarcoplasmic reticulum- (SR-)mediated Ca2+ transport is slower in the fetal heart compared with the adult. Virtually all previous studies of cardiac SR Ca2+ transport were performed in the presence of oxalate, a dicarboxylic anion that is cotransported with Ca2+ in skeletal muscle SR. If anion transport is developmentally regulated in cardiac SR, this could explain, in part, the previously reported results. The purposes of this study were to establish the presence of an SR dicarboxylic anion transport process in the rabbit heart and to determine if the perinatal changes in SR Ca2+ transport occur in a dicarboxylic anion-dependent and/or independent manner. In isolated fetal and adult rabbit cardiac SR membranes, we measured Ca2+ ATPase rates and 45Ca2+ uptake in the presence of the dicarboxylic anions maleate and succinate compared with the zwitterionic buffer PIPES, to which cardiac SR is essentially impermeable. We also measured 14C-succinate uptake by fetal and adult SR membranes. Anion-independent Ca2+ ATPase activity and net 45Ca2+ uptake were significantly lower in the fetal SR membranes than in the adult. Maleate and succinate increased the Ca2+ ATPase rates in the fetal and adult SR, but the effect was significantly greater in the adult. Maleate and succinate stimulated earlier attainment of maximal net Ca2+ uptake in the fetal and adult SR, suggesting that these dicarboxylic anions stimulated the rate of Ca2+ accumulation. Maleate and succinate significantly increased the maximal net Ca2+ uptake in the adult SR, but not in the fetus. The percentage of stimulation of Ca2+ uptake by maleate and succinate was similar in the fetal and adult SR.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental regulation of the sarcoplasmic reticulum calcium pump in the rabbit heart.

Previous studies have demonstrated that myocardial function changes during mammalian perinatal development. The purpose of this study was to evaluate the subcellular basis underlying the slower relaxation in the developing heart by examining perinatal changes in sarcoplasmic reticulum (SR) function, and in SR Ca2+ pump protein and mRNA abundance. We measured Ca2+ uptake and ATPase rates in isolated fetal, newborn, and adult rabbit cardiac SR membranes. In fetal and adult SR membranes, we estimated the active Ca2+ pump protein content by measuring the steady state Ca(2+)-dependent phosphoenzyme content; the total Ca2+ pump protein content was estimated by Western analysis of the immunoreactive Ca2+ pumps. We isolated RNA from fetal and adult hearts and estimated the SR Ca2+ pump mRNA content by Northern analysis. Ca2+ uptake and ATPase rates were significantly lower in the fetal and newborn SR membranes compared with the adult. The contents of active and total Ca2+ pump protein and of Ca2+ pump mRNA were 52-63% lower in the fetus than in the adult. These results indicate that a great deal of the slower sarcoplasmic reticulum Ca2+ uptake and ATPase rates in the fetal rabbit heart can be related to lower Ca2+ pump mRNA and protein contents. It is evident that transcriptional and/or posttranscriptional regulation of the SR Ca2+ pump may form an important part of the subcellular basis of the perinatal change in mammalian cardiac relaxation.

The role of calcium in the energetics of contracting skeletal muscle.

In its second messenger role in skeletal muscle, calcium coordinates the function of muscle (contractile activity) with its overall energetics, thereby controlling the provision of ATP in a time of need. Not only is ATP required for crossbridge turnover in the myofibrils, but it is also needed for the maintenance of ion pumps, nuclear activity, and so forth. When oxygen is limiting, the sustained contractions of both fast and slow muscle (after the immediate burst of activity) is primarily supported by glycogenolysis and the glycolytic pathway (anaerobic). Calcium is important to this process, and the compartmentation of the glycogen particle and some of the enzymes associated with the glycolytic pathway in the terminal cisternae of the sarcoplasmic reticulum ensures that the provision of glucose-6-phosphate to the glycolytic pathway for the generation of the needed ATP proceeds rapidly. The activation of phosphorylase and glycogenolysis by calcium-troponin-C is another example of the tight control of cellular energetics deemed possible by compartmentation within the cell. The regulation by calcium, therefore, is only dependent on the diffusion of calcium rather than diffusion of substrate. When oxygen is not limiting (i.e. when a new steady-state is reached), the aerobic metabolism of pyruvate and fatty acids may be regulated in part by calcium at least in slow skeletal muscle. Oxidative phosphorylation, where ADP is phosphorylated to ATP, is though to be controlled by the concentration of ADP in skeletal muscle. However, because of the obvious compartmentation of the mitochondria within the slow muscle fibre and the higher free calcium required for peak force development (5 mumol/L), the kinetics are theoretically favourable for the calcium cycle in slow muscle mitochondria to play an important role in the regulation of aerobic substrate oxidation, as it does in the heart. Although this hypothesis is attractive based on the available data, the direct demonstration of a major role for calcium as a regulator of substrate oxidation in slow muscle awaits experimentation.