Ablation of posterior atrial ganglionated plexus potentiates sympathetic tachycardia to behavioral stress.

The role of the posterior atrial ganglionated plexus (PAGP) in heart rate (HR) control was tested in unanesthetized dogs (n = 8). Resting HR was unchanged before (85 +/- 20 beats/min, mean +/- SD) versus after (87 +/- 18 beats/min) surgical ablation of these intrinsic cardiac ganglia (PAGPX). However, the peak tachycardia to a 30-s stressful stimulus was significantly increased (P < 0.05) from +53 +/- 22 beats/min before the denervation to +77 +/- 13 beats/min after PAGPX. Conversely, the peak HR increase during the stress after beta-adrenergic blockade was the same before (36 +/- 24 beats/min) versus after (38 +/- 14 beats/min) PAGPX. Moreover, the HR response to a neutral behavioral stimulus, which is mediated primarily by withdrawal of parasympathetic inhibition of the sinoatrial (SA) node, was unaltered by PAGPX. Thus the augmented tachycardia subsequent to PAGPX was attributable primarily to increased sympathetic action at the SA node. These findings indicate that a major role of PAGP parasympathetic neurons is to inhibit sympathoexcitatory effects on HR, probably either via interactions between neurons comprising the intrinsic plexus(es) or perhaps via presynaptic inhibition of sympathetic neurotransmitter release. This organization would allow parasympathetic ganglia within the PAGP to selectively modify sympathetic input to the SA node independent of direct vagal inhibition of pacemaker activity.

Fructose-3-phosphate production and polyol pathway metabolism in diabetic rat hearts.

Previous studies have suggested that polyol-pathway and nonenzymatic glycation may be involved in the development of cardiac myopathy, a well-known manifestation of diabetes. Although the exact etiology of this complication is not fully understood, it is likely to be multifactorial. In this study, we investigated the metabolic consequences of diabetes and the effect of aldose reductase inhibitor (ARI) treatment on cardiac tissues of Sprague-Dawley rats. Perchloric acid (PCA) extracts of hearts from the animals were examined using 31P-nuclear magnetic resonance (NMR), gas chromatography/mass spectrometry (GC/MS), and high-performance liquid chromatography (HPLC). In 31P-NMR spectra of diabetic animals, a peak resonating at the chemical shift of 5.8 ppm with a coupling constant of 10 Hz was identified as fructose-3-phosphate (F3P). Undetectable in controls (< approximately 20 nmol/g), this metabolite was present at a concentration of 81.3 +/- 16.3 nmol/g wet weight (n = 4) in diabetic rat hearts. GC/MS analysis of these extracts from diabetics also identified a decomposition product of F3P, 3-deoxyglucosone (3DG), at a concentration of 9.4 +/- 3.5 nmol/g (n = 3), compared with 0.98 +/- 0.43 nmol/g (n = 3) in controls. No evidence was found for the expected detoxification products of 3-DG, 3-deoxyfructose and 2-keto 3-deoxygluconate. Concomitant with the elevation of F3P and 3DG, fructose and sorbitol levels were also elevated in diabetic animals. Surprisingly, ARI treatment was found to have no effect on the levels of these metabolites. These data suggest that either the heart may be unique in its production of fructose or it may not readily transport the ARI sorbinil. Production of the potent glycating agents F3P and 3DG in diabetics suggests that these compounds may be contributing factors in the glycation of cardiac proteins in the diabetic rat heart.

Identification of galactitol 2-phosphate and galactitol 3-phosphate in the lens of galactose-fed rats.

Production of unusual phosphorylated metabolites in the lens is one of several changes caused by hyperglycemia. Sorbitol 3-phosphate (Sor-3P) and fructose 3-phosphate (Fru-3P) are two such compounds identified in the diabetic lens, and galactitol 2-phosphate (Gal-2P) and galactitol 3-phosphate (Gal-3P) are identified here in the galactosemic lens. These new compounds are the first example of galactitol metabolism in mammalian tissue other than liver. Sor-3P and Fru-3P are also present in the galactosemic lens, apparently synthesized directly from their precursors, sorbitol and fructose, which are elevated in the lens due to increased flux of glucose through the aldose reductase (AR) pathway. The NADPH necessary to support this increased flux is derived from activation of the hexose monophosphate shunt (HMPS), which is clearly demonstrated by a large increase in the concentration of sedoheptulose 7-phosphate (Sed-7P), a HMPS-specific metabolite. Additionally, during 3 weeks of galactose feeding, there is a dramatic increase in lenticular concentrations of galactitol, sorbitol, galactose, and fructose and a sharp decrease in inositol. Glucose remains unchanged. A precipitous loss of both phosphorylated and nonphosphorylated metabolites occurs after 3 weeks, possibly due to lens rupture.

Metabolism of fructose-3-phosphate in the diabetic rat lens.

Fructose-3-phosphate and sorbitol-3-phosphate are produced in diabetic rat lenses by a 3-phosphokinase. While sorbitol-3-phosphate appears to be an inert polyol phosphate, fructose-3-phosphate is a potent cross-linking agent and a potential in vivo source of 3-deoxyglucosone. The objective of this study was to investigate the production and decomposition of fructose-3-phosphate in the diabetic rat lens. The results indicate that this metabolite achieves a steady-state concentration of almost 1 mumol/g wet weight within 2 weeks after the onset of diabetes. These steady-state levels appear to be a consequence of a balance between its production from fructose and its further decomposition to 3-deoxyglucosone. This conclusion is supported by results from disappearance of fructose-3-phosphate in insulin-treated diabetic rats and in vitro incubations of fructose-3-phosphate with amines where production of 3-deoxyglucosone was detected using a number of different methods including mass spectrometry. In agreement with these results, elevated concentrations of 3-deoxyglucosone along with its detoxification product, 3-deoxyfructose, were detected in the diabetic rat lenses. Other sugars and sugar phosphates which were detectable in the diabetic rat lenses were glucose, fructose, glucose-6-phosphate, fructose-6-phosphate, and sedoheptulose-7-phosphate. In conclusion, results from this study suggest that fructose-3-phosphate and 3-deoxyglucosone are likely to be important contributors to the process of nonenzymatic glycation in diabetic rat lenses.

Production of fructose and fructose-3-phosphate in maturing rat lenses.

PURPOSE: A large increase in glycation of crystallins between 1 and 8 months has been demonstrated in lenses obtained from aging rats. The objective of this study was to investigate if an age-associated increase in the levels of any of the phosphorylated and nonphosphorylated sugars in the aging rat lenses could be correlated with this increase. METHODS: Lenses were obtained from Sprague-Dawley rats ranging in age from 2 to 20 months. Trichloroacetic extracts of these tissues were analyzed by using 31P-NMR for sugar phosphates and high-pressure liquid chromatography equipped with an electrochemical detector for sugars and polyols. RESULTS: Although no elevation in the lenticular glucose levels was observed, an age-associated increase in the concentrations of polyol pathway-associated metabolites--sorbitol, fructose, sorbitol-3-phosphate, and fructose-3-phosphate--was detected. In contrast, no significant changes were observed in glycolytic or pentose shunt metabolites. CONCLUSION: Aging lenses accumulate increased concentrations of fructose and fructose-3-phosphate. Because fructose-3-phosphate is a potent glycating agent and a potential in vivo source of 3-deoxyglucosone, its accumulation in the lens, along with fructose, may be a contributing factor in the age-associated increase of nonenzymatic glycation in rat lenses.

Electrophysiological properties of canine cardiac ganglion cell types.

Intracellular recordings were made from 110 canine cardiac ganglion cells to study their electrophysiological properties. According to their discharge responses to depolarizing currents, these neurons were classified as tonic, phasic and non-discharging cells. Of these cell types, the order of the resting membrane potentials was non-discharging > phasic > tonic cells, which was the reverse of the order of their input resistances. Tonic and phasic cells could not be distinguished by the nature of their after hyperpolarizations which involved Ca(2+)-sensitive K+ currents. Although both cell types demonstrated fast Na+ and slow Ca(2+)-mediated action potentials, the tonic cells' action potentials were more sensitive to tetrodotoxin than those of the phasic cells.

Positions of His-64 and a bound water in human carbonic anhydrase II upon binding three structurally related inhibitors.

The 3-dimensional structure of human carbonic anhydrase II (HCAII; EC 4.2.1.1) complexed with 3 structurally related inhibitors, 1a, 1b, and 1c, has been determined by X-ray crystallographic methods. The 3 inhibitors (1a = C8H12N2O4S3) vary only in the length of the substituent on the 4-amino group: 1a, proton; 1b, methyl; and 1c, ethyl. The binding constants (Ki's) for 1a, 1b, and 1c to HCAII are 1.52, 1.88, and 0.37 nM, respectively. These structures were solved to learn if any structural cause could be found for the difference in binding. In the complex with inhibitors 1a and 1b, electron density can be observed for His-64 and a bound water molecule in the native positions. When inhibitor 1c is bound, the side chain attached to the 4-amino group is positioned so that His-64 can only occupy the alternate position and the bound water is absent. While a variety of factors contribute to the observed binding constants, the major reason 1c binds tighter to HCAII than does 1a or 1b appears to be entropy: the increase in entropy when the bound water molecule is released contributes to the increase in binding and overcomes the small penalty for putting the His-64 side chain in a higher energy state.

Sulfonylmethanesulfonamide inhibitors of carbonic anhydrase.

A series of sulfonylmethanesulfonamide derivatives is described, which are inhibitors of carbonic anhydrase (CA). The most potent of these is the racemic fluoro sulfone 9, which inhibits carbon dioxide hydration catalyzed by human CA II (CA-II) with an IC50 of 3 nM. Binding competition studies versus dansylamide indicate that the enantiomers of 9 have different affinities for CA-II, with equilibrium dissociation constants of 3.6 and 0.6 nM. QSAR analysis suggests that the key factors involved in achieving high affinity in this series are sulfonamide acidity, hydrophobicity, and minimization of steric demands at the carbon atom adjacent to the sulfonamide group.

Nicotinic and muscarinic synaptic transmission in canine intracardiac ganglion cells innervating the sinoatrial node.

Nicotinic and muscarinic mediated synaptic mechanisms were investigated in isolated, canine intracardiac ganglia taken from the right atrial fat pad. Using conventional intracellular microelectrode recording techniques on 216 neurons, fast and slow synaptic potentials were evoked by single or trains of stimulation of presynaptic fibers in interganglionic nerves. By varying the stimulus intensity, single or multiple fast excitatory postsynaptic potentials (f-EPSPs) were evoked, indicating the convergence of synaptic inputs on these cells. These f-EPSPs often reached the action potential threshold, were enhanced by the acetylcholinesterase inhibitor physostigmine and were blocked by the nicotinic antagonist hexamethonium. The f-EPSPs were accompanied by a decreased input resistance and had an extrapolated reversal potential of -7.1 mV, suggesting increased conductances to more than one cation. Repetitive presynaptic stimulation evoked slow excitatory postsynaptic potentials (s-EPSPs) in 41% of the cells while slow inhibitory postsynaptic potentials (s-IPSPs) or s-IPSPs followed by s-EPSPs were evoked in 19% of the cells. All slow potentials were abolished by atropine and low Ca2+/high Mg2+ solutions and enhanced by physostigmine. Hexamethonium and adrenergic receptor antagonists had no effects on s-EPSP and s-IPSP. The M1 receptor antagonist pirenzepine reversibly blocked the s-EPSP but not the s-IPSP. On the other hand, the M2 receptor blocker 4-diphenyl-acetoxy-N-methyl piperidine methiodide (4-DAMP) had no effects on the s-EPSP. These observations suggest that s-EPSPs and s-EPSPs are mediated by distinct muscarinic receptors. The amplitude of the s-EPSP and the depolarization evoked by the muscarinic agonist, bethanechol were accompanied by increased input resistance. These responses were decreased in amplitude by membrane hyperpolarization and either reversed polarity or declined to zero amplitude at about -80 mV, suggesting the inhibition of a potassium conductance.

A faculty research and training program for undergraduates in the sciences.

Faculty enthusiasm, with actual hands-on involvement, is a critical factor in establishing student research interest and excitement in a university or college science environment. Such faculty involvement is infectious to students and therefore key to restoring United States leadership in science and technology in the next decades. Most scientists acknowledge that they were initially attracted into scientific careers through one or two notable teachers who served as role models. However, with the introduction of so-called "big science" and its distraction of university faculty away from meaningful, direct student contacts, and with associated withdrawal of funding from "little science" in the college teacher's laboratory, research languishes in nearly all undergraduate teaching institutions. The inspiring college science teacher seems essentially gone, tired or burnt out, unable to keep pace with the rigorous demands of an active research lab while simultaneously meeting the exhausting load of 15-18 (or more) contact teaching hours per week. With all of the associated lecture preparations, student counseling, and Dean's committee assignments, the teacher has little or no scholarly "think time" or opportunity to inspire even the bright students. Without the teacher's honest and evident involvement and deep commitment, the student fails to experience the essential impact of a convincing role model. It is therefore necessary to restore the college science teacher's opportunity and aspirations to be personally involved in research. This can only be accomplished by providing time, facilities, incentives, and encouragement to do what originally attracted the teacher into a career in science and teaching in the first place.(ABSTRACT TRUNCATED AT 250 WORDS)

4-substituted thiophene- and furan-2-sulfonamides as topical carbonic anhydrase inhibitors.

A series of 4-substituted thiophene- and furan-2-sulfonamides was prepared and was found to possess nanomolar-level potency for inhibition of human carbonic anhydrase II in vitro. Selected examples from this group were further evaluated for their potential to act as topically effective ocular hypotensive agents in the ocular normotensive albino rabbit and the ocular alpha-chymotrypsinized rabbit. Solubility studies in water and pH 7.4 buffer were carried out to estimate the ability of compounds to be formulated in solution. The sensitization potential of key representative structures was determined by in vitro glutathione reactivity studies and guinea pig maximization testing.

Thieno[2,3-b]furan-2-sulfonamides as topical carbonic anhydrase inhibitors.

Novel 5-[(alkylamino)methyl]thieno[2,3-b]furan-2-sulfonamides were prepared and evaluated in vitro for inhibition of human carbonic anhydrase II (CA II) and ex vivo for their ability to inhibit Ca II in the albino rabbit eye after topical administration. Compound 11a was found to lower intraocular pressure (IOP) in both the alpha-CT ocular hypertensive albino rabbit and the normal albino rabbit, but was ineffective at lowering IOP in a hypertensive, pigmented monkey model. Since 11a was highly bound to ocular pigment, a series of less basic analogs was prepared. Examples in this series were both less extensively bound to ocular pigment and more active at reducing IOP in pigmented rabbits after topical dosing. Key examples displayed moderate reactivity toward glutathione.

Baroreflex responses to the stress of severe hemorrhage in the rat.

In two groups of anesthetized (sodium pentobarbital), mature Sprague-Dawley rats, 1) aged 2 years and weighing 300-400 grams, 2) aged 6 months weighing 200-300 grams, baroreflex-induced circulatory responses to pressor (graded doses phenylephrine) and depressor (graded doses nitroglycerine) agents were compared to those occurring during progressive hemorrhage in the same animals. Graded withdrawals of blood from the femoral artery elicited progressive hypotension accompanied by bradycardia rather than expected tachycardia. Graded doses of phenylephrine (2.5 ug to 40 ug bolus, via femoral vein) regularly induced elevations in arterial blood pressure with associated reflex bradycardia. Similarly graded doses of nitroglycerine induced a marked decline in arterial blood pressure, without expected tachycardia. As hypotension became more severe (during hemorrhage), atrioventricular conduction slowed and A-V block developed, resulting in statistically greater slowing in ventricular than in atrial excitation and contractile cycles. Heart failure during hemorrhage in the rat is characterized sequentially by severe bradycardia, depressed atrial contractile force, impaired conduction and A-V block, terminating in ventricular, atrial, and finally, in pacemaker failure. Baroreceptor reflexes were blunted or even absent in both young and old animals during induced hypotension.

Epicardial sites for vagal mediation of sinoatrial function.

The posterior atrial fat pad (PAFP) has been described as the probable anatomic location of parasympathetic ganglia mediating sinoatrial (SAN) and atrioventricular nodal function in the mammalian heart. This contrasts with recent localizations of such control elements in the pulmonary vein fat pad (PVFP) and in fatty tissues overlying the junction of inferior vena cava-inferior left atrium (IVC-ILA), respectively. Short bursts (5-8 pulses/burst, 3 bursts/train) of electrical current (1-16 Hz, 400 ms, 1-5 mA) applied directly to the ventral right atrial epicardium via a concentric bipolar electrode (separation 0.3-0.6 mm) during the atrial muscle refractory period, activated subepicardial postganglionic pathways from PVFP and entering the SAN; identical stimulation of dorsal right atrial epicardium between PAFP and SAN excited few or no fiber pathways controlling SAN discharge rate or patterns. In a second series of experiments, injection of a neuronal marker (Fast Blue) into and around SAN, with time (5-10 days) allowed for retrograde transport, resulted in staining of many soma in PVFP but none in IVC-ILA or PAFP. These data strongly affirm the primary, and perhaps exclusive, localization of ganglia that mediate parasympathetic regulation of SAN function in PVFP of the dog's heart, with little or no such participation by ganglia within PAFP or IVC-ILA.

Heart rate control in awake dog after selective SA-nodal parasympathectomy.

Selective surgical sinoatrial (SA)-nodal parasympathectomy (PSX) was used to distinguish the role of the cardiac autonomic nerves in heart rate (HR) control in awake dogs (n = 8) during rest and behavioral arousal. Resting HR increased from 85 +/- 9 beats (mean +/- SE) before surgery to 114 +/- 6 beats after denervation (P less than 0.05). Atrioventricular (AV)-nodal block occurred during the first 1-3 wk post-PSX, but subsequently resolved. Dogs were behaviorally conditioned by following a 30-s tone (CS+) by a 0.5-s shock. Before denervation the CS+ evoked an initial, rapidly developing tachycardia (phase 1), which was followed by a more slowly developing, but larger, phase 2 tachycardia. The selective SA-nodal parasympathectomy essentially abolished the phase 1 conditional HR response (magnitude: 23 +/- 5 vs. 5 +/- 2 beats, pre- vs. postdenervation, respectively). The phase 2 HR increase was similar before and after the denervation (magnitude: 44 +/- 6 vs. 33 +/- 6 beats; rate of increase: 5 +/- 1 vs. 6 +/- 1 beats/s, pre- vs. post-PSX). Beta-Blockade (propranolol, 1 mg/kg) after PSX decreased phase 2 (magnitude: 7 +/- 3 beats; rate of increase: 1 +/- 0.3 beats/s). These data reveal a sterotypic pattern of change in cardiac autonomic nervous drive during a sudden arousal from rest. Phases 1 and 2 appear to be selective and specific indexes of changes in SA-nodal parasympathetic and sympathetic tone, respectively. The selective denervation unmasks during stress a component of HR control that occurs in the absence of adrenergic or cholinergic mechanisms. These data suggest that multiple interactions occur within the intrinsic ganglion plexuses of the heart with respect to HR control.

Gross and microscopic anatomy of the vagal innervation of the rat heart.

Intrinsic cardiac ganglia and their vagal innervation are described from gross and microscopic dissections and functional studies in the anesthetized, open-chest, adult rat. Dissecting microscope sketches of the ventral and dorsal aspects of the rat heart provide gross descriptions of the anatomical course of the vagal cardiac nerves. Histological sectioning of adipose tissue packets surrounding the terminal endings of vagal branches distributed to the roots of the great cardiac vessels (aorta, pulmonary artery, precaval veins) revealed clusters of autonomic ganglia. These packets or "fat pads" were located: (1) along the dorsal surface of the right precava and extending medially toward the aortic root, (2) deep to the aortic arch, (3) in the angle between the root of the left precava and the pulmonary artery on the superior-dorsal surface of the left atrium, and (4) in the rostro-dorsal interatrial septum. Vagal distributions of small terminal branches were traced to each of these pads, which contained numerous autonomic ganglia. Electrical excitation of right or left cervical vagus elicited varying degrees of sinus slowing, slowing of A-V conduction, and suppression in atrial contractile force. Very small quantities (0.5 mg in 10 microliters saline) of the ganglionic blocking agent, hexamethonium (C6) were injected selectively into a single fat pad, followed by repetition of right or left vagal stimulation, with careful analysis of changes in heart rate (paced and unpaced), A-V conduction, and contractile force.

Morphology of intracellularly labeled canine intracardiac ganglion cells.

The purpose of this study was to determine the morphological organization and structure-function correlation of mammalian intracardiac ganglion cells. Conventional intracellular microelectrode techniques were applied to the tissue whole mount preparation of canine intracardiac ganglia. Forty neurons were intracellularly recorded and labeled by means of horseradish peroxidase iontophoresis. Cell morphology was quantitatively analyzed by light microscopy and camera lucida technique. Somata were elongated (mean 62 x 40 microns) and had 2-12 primary dendrites restricted within the ganglion. Almost half of the neurons had either a short axon that was traced only within the ganglion or no axon distinguishable. These neurons may have perhaps been intraganglionically active neurons. The other cells had a long axon that either coursed out of the ganglion to peripheral cardiac tissue or exited the ganglion via interganglionic nerve to innervate more remote cardiac tissue or cells in other intracardiac ganglia. Interaction between neurons was suggested by the close proximity of processes from different neurons. Previously defined electrophysiological cell types (R-, S-, and N-cells), which were significantly different in their passive and active membrane properties, had different morphological features of the somata but not the axonal or dendritic processes. Intraganglionic or long axon neurons were not associated with a particular electrophysiological cell type. These findings provide the possibility of ganglionic modulation of vagal efferent activity in mammalian heart and also provide some morphological basis for the electrophysiological cell types.

Intracellular recording of spontaneous activity of canine intracardiac ganglion cells.

Intracellular recordings were made from 302 canine intracardiac ganglion cells with intact synaptic connections. Fifty-two % of these cells exhibited spontaneous membrane potential changes consisting primarily of miniature excitatory postsynaptic potential (EPSP)-like depolarizations upon which an action potential or burst of action potentials was sometimes evoked. Most of the spontaneous potential changes are presumed to be synaptically mediated involving nicotinic mechanisms because they were blocked by low Ca2+/high Mg2+ and hexamethonium solutions.

Spinal mediation of thermally induced sweating.

The sweat responses of nine patients with physiologically complete lesions of the spinal cord (six cervical and three thoracic) were recorded by two different techniques while the patients were exposed to elevated environmental temperatures. Oral temperatures, heart rate and respiration were monitored throughout the observational periods. Oral temperature invariably rose during exposure to heat and both heart rate and respiration tended to increase. Sweating was detected on all of the test areas by both the iodine-starch-paper technique and the quinizarin technique, but it was of widely varying intensity in different portions of the body. In the patients with cervical lesions sweating was generally profuse on the head and neck and occurred in progressively decreasing intensity down to the level of the umbilicus. It was invariably present, but only in very low intensity, on the lower extremity. Sweating was frequently present as a result of manipulation of the patient during the initial preparations, but this generally declined or stopped before the heat was turned on. With application of heat, sweating was recruited on previously dry areas or increased in intensity on those areas in which it was previously present. After oral temperature had increased moderately, the heat was turned off and the doors of the chamber opened widely so that the heat stimulus was suddenly removed. Despite a continued rise in oral temperature, sweating stopped or decreased dramatically. These results are interpreted to indicate the direct mediation by the isolated spinal cord of reflex sweating responses to a heat stimulus applied to the skin. The general distribution of sweating was similar to that associated with distension of the urinary bladder, and careful attention was taken to avoid this complication. The distribution of sweating on the patients with lesions in the thoracic cord was quite different, being most obvious and profuse on the lower extremities and lower trunk and completely absent from the upper trunk, head and upper extremities.