Daniel L. Cook

Differential Changes of Autonomic Nervous System Function with Age in Man

To assess the relationship between aging and autonomic nervous system function, cardiovascular and pupillary autonomic nervous system reflexes were measured in subgroups of 103 normal male subjects ranging in age from 19 to 82 years (mean age = 39 years). Both the plasma norepinephrine level, a measure of cardiovascular sympathetic nervous system activity, and the mean arterial blood pressure increased with age (r = 0.68 and 0.67, respectively, both p less than 0.001). In contrast, the plasma epinephrine level, a measure of adrenomedullary sympathetic nervous system activity, was unrelated to age (r = 0.08, p = NS). Respiratory variation of heart rate during beta-adrenergic blockade, an index of cardiac parasympathetic nervous system activity, was reduced in older subjects (r = -0.54, p less than 0.001). Thus, there was evidence of an age-related increase of cardiovascular sympathetic nervous system activity and a reduction of cardiac parasympathetic nervous system activity. These findings are consistent with the hypothesis that there is sympathetic nervous system and parasympathetic nervous system compensation of cardiovascular function in response to an age-related decrease in baroreceptor sensitivity. However, dark-adapted pupil size during parasympathetic nervous system blockade, an index of iris sympathetic nervous system activity, declined with age (r = -0.81, p less than 0.001). The latency time for the pupillary response to a light stimulus, an index of iris parasympathetic nervous system activity, was prolonged in older subjects (r = 0.58, p less than 0.001). Thus, both sympathetic nervous system and parasympathetic nervous system inputs to the iris were diminished in older subjects, findings consistent with the generalized decrease of peripheral somatic nerve function that has been reported with aging in man. It is concluded that autonomic nervous system function also declines with aging, but that other age-related changes such as a decline of baroreceptor sensitivity may lead to compensatory autonomic nervous system response, which could mask underlying functional defects.

Quantitative Evaluation of Cardiac Parasympathetic Activity in Normal and Diabetic Man

Heart rate and RR variation (the standard deviation of the mean RR interval for a 5-min period) were evaluated as measurements of cardiac parasympathetic nervous system activity in fasting supine diabetic (N = 22) and comparable age normal (N = 22) subjects. The rate of breathing did not effect heart rate, but was inversely related to the RR variation (r = 0.89, P < 0.01). Heart rate was increased (P < 0.0001) and RR variation decreased (P < 0.05) during β-adrenergic stimulation with isoproterenol and during parasympathetic blockade with atropine (both P < 0.0001). Hence, the cardiac effects of β-adrenergic stimulation may mimic the effects of diminished parasympathetic function. To evaluate parasympathetic control of RR variation, independently of possible effects of increased sympathetic activities, studies were performed during β-adrenergic blockade with propranolol. RR variation during propranolol was less both in 14 diabetic subjects without clinical symptoms of autonomic neuropathy (P < 0.005) and in 8 diabetics with clinical symptoms of autonomic neuropathy (P < 0.001) when compared with 22 age-comparable normal subjects. The measurement of RR variation was very reproducible with a day-to-day coefficient of variation of 9.7 ± 2.8﹪ (x̄ ± SEM) in diabetic subjects with stable hyperglycemia. It is concluded that supine RR variation during a deep respiratory rate and during β-adrenergic blockade is a sensitive, quantitative, and reproducible method to evaluate parasympathetic nervous activity in normal and diabetic subjects. Furthermore, cardiac parasympathetic activity may be diminished in diabetic subjects before clinical symptoms of autonomic neuropathy are evident.

Autonomic Neural Dysfunction in Recently Diagnosed Diabetic Subjects

Because onset of autonomic neural dysfunction in the diabetic syndrome has not been well established, sensitive and quantitative measures of autonomic nervous system (ANS) function were made in 19 non-insulin-dependent (NIDD) and 14 insulin-dependent (IDD) recent-onset diabetic subjects. The known duration of diabetes mellitus in the NIDD subjects was ≤ 12 mo. The duration in the IDD subjects was ≤ 24 mo. RR-variation during beta adrenergic blockade (an index of an ANS reflex involving the cardiac parasympathetic nervous system [PNS] pathway) was smaller than that of control subjects in both NIDD (P < 0.001) and IDD subjects (P < 0.01). This PNS abnormality was not likely to be due to volume depletion since acute volume depletion induced by furosemide in six normal subjects (1608 ± 105 ml, mean ± SEM) did not change RR-variation. Dark-adapted pupil size after topical PNS blockade (an index of iris sympathetic nervous system [SNS] activity) was also smaller in both groups of diabetic subjects (NIDD, P < 0.01; IDD, P < 0.05). Pupillary latency time (an index of an ANS reflex involving iris PNS pathway) was prolonged in the NIDD subjects (P < 0.005) but was not significantly altered in the IDD subjects. Thus, it would appear that the ANS is impaired soon after the diagnosis of diabetes mellitus. We hypothesize that early impairment of the ANS is common in IDD and NIDD subjects. This finding is consistent with the hypothesis that abnormal carbohydrate metabolism is an important factor in the etiology of diabetic autonomic neuropathy.

ATP-Sensitive K+ Channels in Pancreatic β-Cells: Spare-Channel Hypothesis

Since their discovery in pancreatic β-cells, ATP-sensitive K+ channels in the cell membrane have been thought to mediate glucose-induced β-cell depolarization, which is required for triggering the voltage-dependent Ca2+ uptake subserving insulin release. The theory is that metabolism of glucose (and other fuel molecules) increases intracellular ATP or possibly other metabolites that diffuse to the membrane and inhibit the opening of ATP-sensitive K+ channels. This slows the efflux of positively charged K+ and depolarizes the cell. A recurrent source of confusion regarding this idea stems from the early observation that these channels are so exquisitely sensitive to intracellular ATP that channel opening is predicted to be ∼99% inhibited under physiological conditions. To account for this apparent discrepancy, various mechanisms have been proposed that might render the channels less sensitive to intracellular ATP. We use a simple mathematical model to demonstrate that there is no major discrepancy and that, in fact, given the electrophysiological mechanisms existing in the β-cell, the extreme sensitivity of the channels to ATP is appropriate and even mandatory for their physiological function.

Synaptic depression in the localization of sound

Short-term synaptic plasticity, which is common in the central nervous system, may contribute to the signal processing functions of both temporal integration and coincidence detection. For temporal integrators, whose output firng rate depends on a running average of recent synaptic inputs, plasticity modulates input synaptic strength and thus may directly control signalling gain and the function of neural networks. But the firing probability of an ideal coincidence detector would depend on the temporal coincidence of events rather than on the average frequency of synaptic events. Here we have examined a specific case of how synaptic plasticity can affect temporal coincidence detection, by experimentally characterizing synaptic depression at the synapse between neurons in the nucleus magnocellularis and coincidence detection neurons in the nucleus laminaris in the chick auditory brainstem. We combine an empirical description of this depression with a biophysical model of signalling in the nucleus laminaris. The resulting model predicts that synaptic depression provides an adaptive mechanism for preserving interaural time-delay information (a proxy for the location of sound in space) despite the confounding effects of sound-intensity-related information. This mechanism may help nucleus laminaris neurons to pass specific sound localization information to higher processing centres.

ATP-sensitive K+ channels in pancreatic beta-cells. Spare-channel hypothesis

Since their discovery in pancreatic beta-cells, ATP-sensitive K+ channels in the cell membrane have been thought to mediate glucose-induced beta-cell depolarization, which is required for triggering the voltage-dependent Ca2+ uptake subserving insulin release. The theory is that metabolism of glucose (and other fuel molecules) increases intracellular ATP or possibly other metabolites that diffuse to the membrane and inhibit the opening of ATP-sensitive K+ channels. This slows the efflux of positively charged K+ and depolarizes the cell. A recurrent source of confusion regarding this idea stems from the early observation that these channels are so exquisitely sensitive to intracellular ATP that channel opening is predicted to be approximately 99% inhibited under physiological conditions. To account for this apparent discrepancy, various mechanisms have been proposed that might render the channels less sensitive to intracellular ATP. We use a simple mathematical model to demonstrate that there is no major discrepancy and that, in fact, given the electrophysiological mechanisms existing in the beta-cell, the extreme sensitivity of the channels to ATP is appropriate and even mandatory for their physiological function.

Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic β-cells and HIT cells

SummaryATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic β-cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 μm and as high as 500 μm, with maximal activation between 10 and 100 μm ADP in mouse β-cell membrane patches. At concentrations greater than 500 μm, ADP inhibited K(ATP) channels while 10 mm virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mm ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of β-cell membrane potential, increasing ADP in the 10 and 100 μm range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize β-cells.The results suggest that (i) K(ATP)-channel activity is not well predicted by the “ATP/ADP ratio,” and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10–100 μm range as suggested by biochemical studies.

Calcium current inactivation in insulin-secreting cells is mediated by calcium influx and membrane depolarization

Inactivation of voltage-dependent calcium currents was studied in single, dissociated insulin-secreting HIT cells voltage-clamped by the whole-cell patch-clamp method at room temperature. Na and K currents were suppressed by tetrodotoxin, tetraethylammonium, ATP, 4-aminopyridine and Cs. Ca currents activated in less than 10 ms by depolarizations beyond −50 mV from a holding potential of −100 mV and were identified, as in previous studies, by their sensitivity to divalent cation blockade and permeability to Ba as a charge carrier. Sustained depolarization revealed two kinetically distinct phases of inactivation: a rapid phase inactivated approximately 50% of the current in less than 100 ms while the remaining current was inactivated over the next 10–20 s. Rapid inactivation appeared to be due to Ca2+ influx since it was slowed markedly when Ba2+ was used as the current carrier, while the degree of inactivation mercased and decreased with increasing depolarization in direct parallel with the U-shaped current-voltage relationship for inward Ca current. Slow inactivation appeared to be voltage-dependent since current could be inactivated (by ≈20%) by 10 s long depolarizations to potentials below the threshold for activating Ca current, slow time constants of inactivation were voltage-dependent and slow inactivation persisted when Ca was replaced with Ba. Ca currents with low activation thresholds (in the −50 to −30 mV range) appeared to be preferentially inactivated by the rapid Ca-dependent mechanism. Recovery of slowly inactivated Ca current was very slow and currents inactivated by larger depolarizations required longer recovery time than those elicited by smaller depolarizations. Rapid and slow inactivation mechanisms may be important in understanding the fast spiking and slow plateau depolarizations seen in pancreatic B-cells exposed to stimulatory levels of glucose.

Voltage-gated Ca2+ current in pancreatic B-cells

Voltage-dependent inward Ba++ and Ca++ currents were recorded in cultured neonatal rat pancreatic islet cells using the whole-cell voltage clamp technique. Outward current was suppressed by internal Cs+ and ATP and external TEA. Inward currents activated rapidly and decayed to a variable extent. The current decay was particularly marked when using long duration or large depolarizing pulses. Currents were due to Ca++ channel activation since they were abolished by omitting Ba++ and Ca++ or including Co++.

Pancreatic B cells are bursting, but how?

Insulin secretogogues have long been known to stimulate and modulate bursting electrical activity in pancreatic islet B cells and thereby supply extracellular Ca2+ for the exocytosis of insulin. Recent results have ruled out a long-held hypothesis for the mechanism of burst formation that postulated key roles for intracellular Ca2+ accumulation and activation of Ca(2+)-activated K+ channels. Here, we present an alternative hypotheses based on a persistent Ca2+ conductance and, possibly, phasic activation of ATP-sensitive K+ channels. These hypotheses are compared with mechanisms of bursting proposed for invertebrate and mammalian neurons.