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Most visceral organs are under the control of the autonomic nervous system (ANS). Information on the state and function of these organs is constantly relayed to the central nervous system (CNS) by sensory afferent fibers. The CNS integrates the sensory inputs and sends neural commands back to the organ through the ANS. The autonomic ganglia are the final site for the integration of the message traveling from the CNS. Nicotinic acetylcholine receptors (nAChRs) are the main mediators of fast synaptic transmission in ganglia, and therefore, are key molecules for the processing of neural information in the ANS. This review focuses on the role of nAChRs in the control of organ systems such as heart, gut, and bladder. The autonomic control of these organ systems is discussed in the light of the results obtained from the analysis of mice carrying mutations targeted to nAChR subunits expressed in the ANS., (Copyright 2002 Wiley Periodicals, Inc.)

Corticotropin-releasing hormone (Crh) plays an important role in modulating physiological and behavioral responses to stress. Its actions are mediated through two receptors, Crhr1 and Crhr2. Urocortin (Ucn), a Crh-related neuropeptide and the postulated endogenous ligand for Crhr2, is a potential mediator of stress responses. We generated Ucn-deficient mice using embryonic stem cell technology to determine its role in stress-induced behavioral and autonomic responses. Unlike Crhr1- or Crhr2-deficient mice, Ucn-deficient mice exhibit normal anxiety-like behavior as well as autonomic regulation in response to stress. However, the mutant mice display an impaired acoustic startle response that is not due to an obvious hearing defect. Thus, our results suggest that Ucn does not play an essential role in stress-induced behavioral and autonomic responses. Ucn may modulate the acoustic startle response through the Ucn-expressing neuron projections from the region of the Edinger-Westphal nucleus.

Nicotinic acetylcholine receptors (nAChRs) have been implicated in Alzheimer's disease, Parkinson's disease, epilepsy, nicotine addiction, schizophrenia, and autonomic dysfunctions. Although nicotine may be used therapeutically either alone or in association with other drugs, its beneficial effects are limited by its addictive properties and a number of other side effects. A deeper understanding of nicotinic cholinergic mechanisms is necessary to develop nAChR ligands that are more selective, less toxic, and more therapeutically effective than nicotine. Although there has been significant progress identifying the nAChR subunits that form functional nAChRs, there is limited information associating the location and function of nAChR subtypes in the nervous system. Several groups have genetically engineered mice in which one or more genes encoding nAChR subunits has been deleted or altered. Mice with nAChR mutations targeted to subunits that are highly expressed in the CNS have brought insight into the nAChR mechanisms involved in nicotine addiction, analgesia, aging, and nicotine-induced behaviors. Mutations targeted to nAChR subunits that are highly expressed in the peripheral nervous system have opened a window on the complex mechanisms governing autonomic control of peripheral organs. This review examines nAChRs in the autonomic control of peripheral organ systems as gleaned from studies of nAChR mutant mice.

High doses of nicotine, the addictive component of tobacco, induce clonic-tonic seizures in animals. Pharmacological and biochemical data have suggested that alpha 7-containing neuronal nicotinic receptors (nAChRs) contribute to these seizures. To study potential alpha 7 contributions, we examined alpha 7 subunits with a Leu250-to-Thr substitution in the channel domain, which creates a gain-of-function mutation. Previous studies have shown that mice homozygous for the alpha 7 L250T mutation (T/T) die shortly after birth, but animals heterozygous for the mutation (+/T) are viable and grow to adulthood. Hippocampal neurons from the +/T mice exhibited altered alpha 7-type currents with increased amplitudes and slower desensitization kinetics, confirming a partial gain of function for the alpha 7 nAChR. We found that +/T mice were more sensitive to the convulsant effects of nicotine compared with their wild-type (+/+) littermates. Furthermore, although their behavior was normal in basal conditions, +/T mice showed a unique nicotine-induced phenotype, consisting of head-bobbing and paw-tapping movements. Increased sensitivity to nicotine-induced seizures occurred despite a 60% decline in brain alpha 7 nAChR protein levels. There were no changes in the levels of alpha 4, alpha 5, alpha 6, alpha 7, beta 2, and beta 4 mRNA, or in [(125)I]epibatidine and [(3)H]nicotine binding between +/T and +/+ mice. Recent data from our laboratory show that alpha 7-null mice maintain normal sensitivity to nicotine-induced seizures. Hence, these present findings suggest that alterations in the properties rather than absence of alpha 7 nAChRs might affect the mechanisms underlying the convulsive properties of nicotine.

Nicotine is the primary addictive component in tobacco, and at relatively low doses it affects cardiovascular responses, locomotor activity, thermoregulation, learning, memory, and attention. At higher doses nicotine produces seizures. The mechanisms underlying the convulsive effects of nicotine are not known, but studies conducted on a number of inbred strains of mice have indicated a positive correlation between the number of alpha-bungarotoxin (alpha-BTX) binding sites in the hippocampus and the sensitivity to nicotine-induced seizures. Because alpha7-containing neuronal nicotinic acetylcholine receptors (nAChRs) represent the major binding site for alpha-BTX, mice lacking the alpha7 nAChR subunit were predicted to be less sensitive to the convulsive effects of nicotine. To test this hypothesis, we injected nicotine intraperitoneally in alpha7 mutant mice and found that the dose-response curve for nicotine-induced seizures was similar in the alpha7 +/+, alpha7 +/- and alpha7 -/- mice. The retained sensitivity to the convulsant effects of nicotine could not be explained by the presence of cholinergic compensatory mechanisms such as increases in mRNA levels for other nAChR subunits, or changes in binding levels or affinity for nicotinic ligands such as epibatidine and nicotine. These findings indicate that alpha7 may not be necessary for the mechanisms underlying nicotine-induced seizures.

In developed countries, tobacco use is estimated to be the largest single cause of premature death [Lancet 339 (1992) 1268]. Nicotine is the main addictive component of tobacco that motivates continued use despite the harmful effects. Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the mammalian central nervous system (CNS), where they normally respond to acetylcholine (ACh) and modulate neuronal excitability and synaptic communication. Nicotinic receptors are structurally diverse and have varied roles. Presynaptic and preterminal nAChRs enhance neurotransmitter release. Postsynaptic and somal nAChRs mediate a small proportion of fast excitatory transmission and modulate cytoplasmic second messenger systems. Although the impact of nicotine obtained from tobacco is not completely understood, a portion of nicotine's addictive power is attributable to actions upon the dopaminergic systems, which normally help to reinforce rewarding behaviors. As obtained from tobacco, nicotine activates and desensitizes nAChRs, and both processes contribute to the cellular events that underlie nicotine addiction.

The autonomic nervous system controls and coordinates several cardiovascular functions, including heart rate, arterial pressure, blood flow and vasomotor tone. Neuronal nicotinic acetylcholine receptors (nAChRs) are the interface between the nervous system and the cardiovascular system, but it is not known which nAChR subtypes regulate autonomic function in vivo. Nicotinic AChRs containing the alpha7 subunit are a candidate subtype in autonomic ganglia. Stimulation of these nAChRs can increase neurotransmitter release via presynaptic mechanisms, as well as mediate fast synaptic transmission via postsynaptic mechanisms. To investigate the role of the alpha7 nAChR subunit in cardiac autonomic function, we measured baroreflex-mediated responses in alpha7 null mice. Here we show that the alpha7 null mice have impaired sympathetic responses to vasodilatation, as sodium nitroprusside infusion triggered a 48% heart rate increase in wild type mice but only a 21% increase in the alpha7 nulls (P < 0.001). The mutant mice developed supersensitivity to adrenergic agonists, although norepinephrine release from sympathetic nerve terminals could be elicited through mechanisms alternative to nAChR stimulation. Baroreflex-mediated parasympathetic responses were normal in alpha7 null mice. The decreased baroreflex-mediated tachycardia in alpha7 mutant mice indicates that alpha7-containing nAChRs participate in the autonomic reflex that maintains blood pressure homeostasis. The alpha7 mutant mice may serve as a model of baroreflex impairment arising from autonomic dysfunction.

Micturition is achieved through complex neurological mechanisms involving somatic, autonomic and central components. This article briefly reviews recent findings on the autonomic control of urinary bladder function. Neuronal nicotinic acetylcholine receptors mediate fast synaptic transmission in autonomic ganglia, and activation of nicotinic receptors in parasympathetic bladder neurons produces contraction of the detrusor muscle. Autonomic ganglia contain transcripts for the alpha(3), alpha(4), alpha(5), alpha(7), beta(2) and beta(4) nicotinic subunits, which can assemble to form multiple nicotinic receptor subtypes, but the exact nicotinic receptor subunit composition in bladder ganglia is unknown. Mutant mice lacking the alpha(3) or the beta(2) and the beta(4) nicotinic subunits have enlarged bladders with dribbling urination and develop urinary infection and bladder stones. Bladder strips from alpha(3) null mice do not respond to nicotine but contract when stimulated with a muscarinic agonist or electric field stimulation. Mice lacking the beta(2) subunit have no overt bladder phenotype, and their bladders contract in response to nicotine. Surprisingly, bladder strips from beta(4) mutant mice do not respond to nicotine despite the absence of major bladder dysfunction in vivo. These findings suggest that nicotinic receptors containing the alpha(3) and the beta(4) subunits are necessary for normal bladder function.

Transcripts for the beta2 and the beta4 nicotinic acetylcholine receptor (nAChR) subunits are found throughout the CNS and the peripheral nervous system. These two beta subunits can form heteromultimeric channels with any of the alpha2, alpha3, alpha4, or alpha5 subunits in heterologous expression systems. Nonetheless, the subunit composition of native nAChRs and the role of different nAChR subtypes in vivo remain unclear. We prepared null mutations for the beta2 and the beta4 genes and bred beta2-/-beta4-/- mice by mating mice of identical beta2-/-beta4+/- or beta2+/-beta4-/- genotype. The beta2-/- and the beta4-/- single-mutant mice grow to adulthood with no visible phenotypic abnormalities. The beta2-/-beta4-/- double mutants survive to birth but have impaired growth and increased perinatal mortality. They also present enlarged bladders with dribbling urination and develop urinary infection and bladder stones. The ocular pupils are widely dilated and do not constrict in response to light. Histological studies revealed no significant abnormalities of brain or peripheral tissues except for hyperplasia in the bladder mucosa of beta4-/- and beta2-/-beta4-/- mutants. Bladder strips from beta2-/-beta4-/- mice did not respond to nicotine but contracted when stimulated with a muscarinic agonist or electric field stimulation. Bladder strips from beta4 mutants did not respond to nicotine despite the absence of major bladder dysfunction in vivo. Acetylcholine-activated whole-cell currents were absent in superior cervical ganglion neurons from beta2-/-beta4-/- mice and reduced in neurons from beta4-/- mice. Although there is apparent redundancy and a superficially normal phenotype in beta2-/- and beta4-/- mice, physiological studies indicate major deficits in the beta4-/- mice. Our previous description of a similar phenotype in alpha3-/- mice and the current data suggest that the alpha3 and the beta4 subunits are major components in autonomic nAChRs. The phenotype of the beta2-/-beta4-/- and alpha3-/- mice resembles the autosomal recessive megacystis-microcolon-hypoperistalsis syndrome in humans.

The alpha3 subunit of the neuronal nicotinic acetylcholine receptor is widely expressed in autonomic ganglia and in some parts of the brain. The alpha3 subunit can form heteromultimeric ion channels with other alpha subunits and with beta2 and beta4 subunits, but its function in vivo is poorly understood. We prepared a null mutation for the alpha3 gene by deletion of exon 5 and found that homozygous (-/-) mice lacked detectable mRNA on Northern blotting. The -/- mice survive to birth but have impaired growth and increased mortality before and after weaning. The -/- mice have extreme bladder enlargement, dribbling urination, bladder infection, urinary stones, and widely dilated ocular pupils that do not contract in response to light. Detailed histological studies of -/- mice revealed no significant abnormalities in brain or peripheral tissues except urinary bladder, where inflammation was prominent. Ganglion cells and axons were present in bladder and bowel. Bladder strips from -/- mice failed to contract in response to 0.1 mM nicotine, but did contract in response to electrical field stimulation or carbamoylcholine. The number of acetylcholine-activated single-channel currents was severely reduced in the neurons of superior cervical ganglia in -/- mice with five physiologically distinguishable nicotinic acetylcholine receptor subtypes with different conductance and kinetic properties in wild-type mice, all of which were reduced in -/- mice. The findings in the alpha3-null mice suggest that this subunit is an essential component of the nicotinic receptors mediating normal function of the autonomic nervous system. The phenotype in -/- mice may be similar to the rare human genetic disorder of megacystis-microcolon-intestinal hypoperistalsis syndrome.

The alpha7 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) is abundantly expressed in hippocampus and is implicated in modulating neurotransmitter release and in binding alpha-bungarotoxin (alpha-BGT). A null mutation for the alpha7 subunit was prepared by deleting the last three exons of the gene. Mice homozygous for the null mutation lack detectable mRNA, but the mice are viable and anatomically normal. Neuropathological examination of the brain revealed normal structure and cell layering, including normal cortical barrel fields; histochemical assessment of the hippocampus was also normal. Autoradiography with [3H]nicotine revealed no detectable abnormalities of high-affinity nicotine binding sites, but there was an absence of high-affinity [125I]alpha-BGT sites. Null mice also lack rapidly desensitizing, methyllycaconitine-sensitive, nicotinic currents that are present in hippocampal neurons. The results of this study indicate that the alpha-BGT binding sites are equivalent to the alpha7-containing nAChRs that mediate fast, desensitizing nicotinic currents in the hippocampus. These mice demonstrate that the alpha7 subunit is not essential for normal development or for apparently normal neurological function, but the mice may prove to have subtle phenotypic abnormalities and will be valuable in defining the functional role of this gene product in vivo.

In an attempt to explore the relationships between conformation of chemotactic peptides related to elastin and their biological activity we have studied five peptides: VGVAPG, VGVPG, VGAPG, GVAPG and GGVPG in solvents of different polarities which may mimic the environmental conditions at the receptor site. CD and NMR studies showed that GVAPG has no preference for structured conformations, while the other peptides may assume folded conformations in organic solvents. All these peptides but GGVPG showed chemotactic activity for monocytes. The chemotactic activity of VGVPG, VGAPG and VGVAPG was inhibited by lactose, while chemotaxis of peptide GVAPG was insensitive to lactose, suggesting the existence of different chemotactic receptors.

We report the cloning and functional expression of a novel K+ channel beta-subunit from human atrium, hKv beta 3. hKv beta 3 is highly homologous to the two beta-subunits cloned from rat brain, Kv beta 1 and Kv beta 2, but has an essentially unique stretch of 79 N-terminal residues. Upon expression in Xenopus oocytes, hKv beta 3 accelerates the inactivation of co-injected hKv1.4 currents and induces fast inactivation of non-inactivating co-injected hKv1.5 currents. By contrast, hKv beta 3 had no effect on hKv1.1, hKv1.2, or hKv2.1 currents. Thus, hKv beta 3 represents a third type of K+ channel beta-subunit which modulates the kinetics of a unique subset of channels in the Kv1 subfamily.

The cardiac inward rectifier current (IK1) contributes to the shape and duration of the cardiac action potential and helps to set the resting membrane potential. Although several inwardly rectifying K+ channels (IRKs) from different tissues have been cloned recently, the nature and number of K+ channels contributing to the cardiac IK1 are presently unknown. To address this issue in human heart, we have used the reverse-transcriptase-polymerase chain reaction (PCR) technique with human atrial total RNA as a template to identify two sequences expressed in heart that are homologous to previously cloned IRKs. One of the PCR products we obtained was virtually identical to IRK1 (cloned from a mouse macrophage cell line); the other, which we named hIRK, exhibited < 70% identity to IRK1. A full-length clone encoding hIRK was isolated from a human atrial cDNA library and functionally expressed in Xenopus oocytes. This channel, like IRK1, exhibited strong inward rectification and was blocked by divalent cations. However, hIRK differed from IRK1 at the single-channel level: hIRK had a single-channel conductance of 36 pS compared with 21 pS for IRK1. We have identified single channels of 41, 35, 21, and 9 pS in recordings from dispersed human atrial myocytes. However, none of these atrial inward rectifiers exhibited single-channel properties exactly like those of cloned hIRK expressed in oocytes. Our findings suggest that the cardiac IK1 in human atrial myocytes is composed of multiple inwardly rectifying channels distinguishable on the basis of single-channel conductance, each of which may be the product of a different gene.

YGVG and GLVPG, two new chemokinetic peptides, were identified in elastolytic digests of elastin, besides the known chemoattractant hexapeptide VGVAPG. In order to identify possible sequences responsible for the chemotactic and chemokinetic activities and to obtain structure-activity relationships we synthesized some analogues of these peptides: FGVG (an analogue of YGVG), GVAPG and VGAPG (derived from the hexapeptide by deletion of Val1 or Val3). FGVG has a higher chemotactic activity than YGVG (chemotactic indices of 0.62 and 0.49, respectively, at 10(-11) M) and is both chemotactic and chemokinetic. Checkerboard analysis demonstrated that both peptides derived from the hexapeptide present, in addition to the chemotactic activity, a chemokinetic activity. The chemotactic index of GVAPG is 0.66 at 10(-10) M, while for VGAPG it is 0.86 at 10(-9) M. These results indicate that the deletion of the N-terminal residue of the elastin chemotactic peptides, VGVAPG and GFGVG, gives rise to chemokinetic activity. CD and NMR studies showed that all peptides are largely unordered in aqueous solution.

We investigated serum and aortic tissue lipid content, in vitro aortic response to drugs, and morphology of thoracic aorta in Pittsburgh Yoshida rats (YOS), a new animal model of endogenous hyperlipidemia. Experiments were performed on 2-, 6-, and 18-month-old rats. Normolipidemic Brown Norway rats (BN) were used as controls. Both serum cholesterol and triglycerides increased significantly with age in YOS rats, but remained constantly low in the control group. In YOS rats, absolute serum concentration of high density lipoprotein (HDL)-cholesterol increased significantly with age, although HDL-cholesterol/total-cholesterol ratio decreased. In contrast, no difference in cholesterol content in aortic tissue was detected between the two animal strains or among different age groups. The contractile force generation of thoracic aorta to norepinephrine (NE) and serotonin increased with age in both strains of animals. The endothelium-dependent relaxation induced by acetylcholine (ACh) was significantly reduced in 6- and 18-month-old YOS as compared with 2-month-old YOS but not in BN. ATP-induced relaxation was significantly impaired in YOS thoracic aorta. In contrast, the relaxation induced by NaNO2 acting in smooth muscle did not vary with age in either YOS or BN. Only alterations in endothelial cells, not typical atheromatous injuries in thoracic aorta wall were detected in YOS even at age 18 months. Our data indicate that despite high serum lipid levels, YOS do not develop typical atheromatous lesions or functional and morphologic damage of smooth muscle cells in thoracic aorta, whereas YOS show decreased endothelium-dependent relaxation and morphologic alteration of endothelial cells.

The pore of a chimeric K+ channel, CHM, differed from its parental host channel, Kv2.1, by 9 amino acids. Four were located in a putative deep region and 5 in a nearby outer mouth. Point reversions were without restorative effects, and reversions V369I or L374V in the deep pore produced novel phenotypes. Among double mutations, only V369I and L374V were effective in restoring the Kv2.1 pore phenotype. Adding a change in charge at Q382K in the outer pore fully restored the parental phenotype. Thus, the pore appears to have an inner, deep region where ions such as K+ and TEA+ may be regulated by nonpolar residues and an outer region where ions may be regulated by charged residues.

The aim of this study was to evaluate the effects of aging on endothelium-dependent and endothelium-independent relaxation of rabbit thoracic aorta from New Zealand white rabbits aged 4-6 and 7-12 months. The contractile response to noradrenaline (NA) decreased with increasing age, but NA [EC50] did not vary significantly. Acetylcholine (Ach)-induced relaxation of aortic rings precontracted with NA [EC50] did not change significantly with increasing age. The relaxation induced by ATP of aortic rings, precontracted with NA [EC50], was significantly greater in young than in adult rabbits. This difference between young and adult animals became more evident in aortic rings deprived of endothelium: in adult animals, the ATP-induced relaxation of aortic rings with endothelium was significantly greater than in the rings without endothelium. The endothelium-independent relaxation by sodium nitrite (NaNO2) at lower concentrations was significantly greater in young than in adult rabbit aortic rings precontracted with NA [EC50]. Concluding, the age-induced changes in vascular response in male New Zealand white rabbits are related to an impaired mechanism at smooth muscle level.

Peroxidase isozymes from winter wheat (Triticum aestivum L. cv Orso) seedlings extracts showed phenoloxidase-like activity, becoming visible on polyacrylamide gels also in the absence of hydrogen peroxide. The results obtained after a characterization of the two activities, based on their substrate specificity, on their selective inhibition, and on the possible occurrence of artifacts, suggested the existence of polyfunctional peroxidase isozymes. Different isozymes possessing only phenol oxidase activity were not found in the same plant material. This appears to be the first evidence of phenoloxidase-acting isoperoxidases in winter wheat.

Male New Zealand rabbits were fed a high cholesterol (1%) diet for 8 weeks followed by a normal diet for 8 additional weeks. Rabbits on normal diet were used as controls. Serum cholesterol, elevated at the end of the cholesterol feeding period, returned to normal at the time of observation. Aortic wall lesions were severe and vessel wall cholesterol in the thoracic aorta was approximately 10 times the level in control animals. In vitro, thoracic aortic rings were used to measure contractile response to noradrenaline (NOR); no difference in response was found between normal and treated rabbits. Rings pre-contracted with NOR, were exposed to acetylcholine (Ach), ATP and NaNO2. Proximal and distal rings from normal animals showed a dose-dependent relaxation response to all agents, though maximal dilation occurred at an intermediate concentration of Ach. The rings from treated rabbits relaxed normally in response to NaNO2, but ATP relaxation was reduced in proximal and distal rings and Ach induced a contractile response. HPLC analysis of tissue extracts from aortic arch and distal thoracic aorta showed reduced ATP, ADP, GTP and GDP, and increased AMP. These changes resulted in a reduced energy charge and a reduction in total adenine nucleotides. The data indicate that cholesterol feeding, followed by normal diet, causes severe alterations of aortic vessel wall and that at an advanced stage of the diet-induced experimental disease energy metabolism and endothelium dependent vasodilation remain impaired even in the presence of normal serum lipids.

1. (-)-N6-phenylisopropyladenosine (R-PIA) and N6-cyclohexyladenosine (CHA), highly selective agonists at A1-adenosine receptors, 5'-N-ethyl-carboxamidoadenosine (NECA), a non-selective agonist at A1 and A2 receptors, and 2-phenylaminoadenosine (CV-1808), a selective A2 agonist, were compared in spontaneously beating and electrically driven atria. R-PIA, CHA and NECA inhibited contraction in both preparations. CV-1808 was not effective up to 500 nM. 2. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a new selective A1 receptor antagonist, competitively inhibited the effects of the adenosine agonists, at low concentrations (IC50 less than 1 nM). 3. CHA and NECA were able to inhibit the positive inotropic effect of Bay K 8644 both in spontaneously beating and in electrically driven atria. 4. R-PIA, CHA and NECA (agonists), 8-phenyltheophylline (PT) and DPCPX (antagonists), failed to influence [3H]-nitrendipine binding on microsomal membranes from guinea-pig atria and ventricles in a range of concentrations from 1 nM to 100 microM. 5. The data support the existence of A1 receptors in atrial tissue. No evidence for a direct interaction between adenosine analogues and Bay K 8644 was found at the level of slow calcium channels. Adenosine analogues appear to antagonize the effects of Bay K 8644 indirectly by activation of A1 receptors.

Three sets of parameters, (i) relaxation to acetylcholine (Ach), ATP and NaNO2, (ii) cholesterol content in aortic tissue, and (iii) energy metabolism were compared in normal and atherosclerotic rabbits, fed 1% cholesterol for eight weeks. A special protocol was envisaged to permit a strict comparison between Ach, ATP and NaN O2 at different levels of thoracic aorta, in each rabbit. A gradual impairment of the endothelium-dependent relaxation to Ach and ATP was found at different levels of the thoracic aorta from hypercholesteromic rabbits. By contrast, NaNO2--endothelium-independent--maintained its relaxing power quite normally at all aortic levels. A close correlation was evident between the impairment of aorta relaxation to Ach and the cholesterol infiltration in the vessel wall, being the correlation coefficient -0.85 (P less than 0.001). A correlation was also evident for ATP, but to a lower degree, being the correlation coefficient -0.61 (P less than 0.01). Energy metabolism and related parameters (ATP, ADP, AMP, adenosine, inosine, GTP, GDP, guanosine, NAD, NADP, total adenylate nucleotides and adenylate energy charge) were not modified by the cholesterol diet. These data show that the gradual impairment of endothelium-dependent relaxation, decreasing down the thoracic aorta of hypercholesterolemic rabbits, and correlated with cholesterol content in the aortic wall, may be considered as an index of a very early atherogenic damage, prior to variation in the parameters of energy metabolism and purine turnover.

1. The effect of three different potassium channel blockers (tetraethylammonium, TEA; 4-aminopyridine, 4-AP; and apamin) and of variations in the concentration of K+ and Ca2+ in the medium, have been studied on the responses of guinea-pig isolated atria to (-)-N6-phenylisopropyladenosine (R-PIA), a stable adenosine A1-receptor agonist, and to carbachol, a muscarinic agonist. R-PIA and carbachol showed the same negative inotropic effects over a similar range of concentrations (3-300 microM), both in spontaneously beating and in electrically driven atria. 2. TEA (0.1 to 20 mM) and 4-AP (0.3 to 3 mM), both antagonized the negative inotropic and chronotropic effects of carbachol in a concentration-dependent manner. In contrast, these compounds failed to inhibit the effects induced by R-PIA. Apamin, a specific blocker of a low conductance Ca2+-activated K+ channel, was ineffective in accordance with the absence of these channels in atrial tissue. 3. TEA (0.1 to 20mM) inhibited the negative inotropic effect of carbachol, but not that of R-PIA, in atria paced and depolarized by a high K+ medium (22 mM). In this preparation Na+ current is abolished and the contraction induced by noradrenaline and electrical stimulation is solely dependent on Ca2+ influx currents. 4. Stepwise addition of Ca2+ to a calcium-depleted perfusing medium of electrically driven atria, induced a positive inotropic effect which was inhibited by R-PIA. In contrast, carbachol had no effect. 5. In agreement with our previous study, the data suggest that R-PIA acts on isolated atria by inhibiting Ca2+ influx through L-channels.