Journal of the autonomic nervous system最新文献

During physical activity, there is a graded withdrawal of vagal cardiac tone and a graded increase in sympathetic cardiac and vasomotor tone, initiated through both central command from the somatic motor cortex and muscle chemoreceptive and mechanoreceptive inputs. In parallel, there is an upward resetting of the operating point of the arterial baroreflex, with preserved reflex sensitivity. In contrast to the traditional interpretation that blood flow through exercising muscle is independent of vasomotor neural influences because of the dominance of local dilator metabolites, recent evidence suggests that both constrictor and dilator sympathetic neural influences may be involved in determining absolute levels of perfusion. Post-exercise, there is a period of relative hypotension that is associated with decreased peripheral resistance. Some, but not all, evidence indicates a causal role for reduced sympathetic drive. Chronic exercise training appears to reduce resting sympathetic activity, with parallel changes in the gain of a variety of cardiovascular autonomic reflexes initiated from cardiovascular sites. These changes may be attributable at least partly to masking of arterial baroreflexes by the impact of elevated blood volume on low-pressure baroreceptors. The reductions in sympathetic drive that follow training are more pronounced in patients with essential hypertension than in normotensive individuals and are likely to underlie the anti-hypertensive effect of exercise.

P2X receptors are ATP-gated cation channels; they form as homomers or heteromers from a family of seven related subunits. In particular, heteromeric channels comprising P2X₂ and P2X₃ subunits, or P2X₁ and P2X₅ subunits, show distinctive physiological and pharmacological properties in heterologous expression systems. There is substantial evidence that one of the native P2X receptors in sensory neurones corresponds to the P2X_2/3 heteromer, but there is no evidence for P2X_1/5 heteromers in native tissue. We recorded currents in response to activation of heteromeric P2X_1/5 receptors expressed in HEK293 cells to characterize further their functional properties. The ATP concentration–response curve had a threshold concentration of 1 nM, and a Hill slope of one. TNP-ATP was a weak partial agonist, and a non-competitive antagonist which inhibited maximal ATP currents by 60%. Increasing or decreasing pH from 7.3 shifted the ATP concentration–response curves to the right by fivefold and decreased the maximum current by 40%. Calcium permeability was lower than that observed for other P2X receptors (P_Ca/P_Na ratio=1.1). The nanomolar sensitivity of this receptor revealed a steady release of ATP from HEK293 cells, providing an extracellular concentration which ranged from 3 to 300 nM. Noradrenaline (0.3–30 μM) increased ATP-evoked currents by 35%; this facilitation occurred within 20 ms. We also recorded excitatory junction potentials (EJPs) from guinea-pig submucosal arterioles. EJPs were inhibited by suramin and PPADS (IC₅₀s of 0.2 μM and 20 μM) but TNP-ATP (0.1–10 μM) inhibited EJPs by <30%. Noradrenaline (0.3–30 μM in the presence of phentolamine and propranolol) decreased EJPs in control preparations but facilitated EJPs by 5–20% in submucosal arterioles from reserpinized guinea-pigs. These properties are discussed in relation to P2X receptors underlying EJPs at autonomic neuroeffector junctions.

I have had the pleasure and privilege of being involved in one facet of Geoffrey Burnstock’s early career. I have reviewed this work together with more recent developments in the area. In 1968, the presence of non-adrenergic, non-cholinergic inhibitory nerves had been established but the identity of their neurotransmitter was unknown. Stimulation of these nerves in recycled perfused toad and guinea-pig stomachs caused release of adenosine and inosine. When ATP was added to recycled perfusates, it was broken down to adenosine and inosine. These findings together with information that AMP was released from stimulated, isolated turkey Auerbach’s plexus which was known to contain the nerves, suggested that ATP could be the neurotransmitter. This was supported by observations that ATP elicited responses similar to that of nerve stimulation in a variety of tissues. Developments from the early purinergic nerve hypothesis are considered including independence of extracellular actions of ATP from its intracellular actions, identification and cloning of purinoceptors and cotransmission of ATP with other substances.

In this report, the regulatible expression by tetracycline of the rat recombinant P2X₃ receptor in stably transfected Chinese hamster ovary (CHO-K1) expressing the tetracycline-controlled transactivator (tTA) is described. cDNA encoding the rat P2X₃-receptor was subcloned into pTRE (a tetracycline-repressible expression vector) which was used to transfect stably CHO-K1 tTA cells. Using whole cell patch clamp techniques, 100 μM ATP evoked inward currents of 2.9±1.6 nA in transfected cells grown in the absence of tetracycline (tet−). The P2X₃ receptor protein was detectable by immunoblot as early as 24 h and protein expression levels continued to increase as much as 192 h following activation of tTA by the removal of the antibiotic. Saturation binding isotherms using [³⁵S]ATPγS yielded a pK_d of 8.2±0.1 and a B_max of 31.9±3.5 pmol/mg protein in tet− cell membranes and a pK_d of 8.1±0.1 and a B_max of 5.8±0.8 pmol/mg protein in tet+ cell membranes. The agonist ligands 2MeSATP and αβMeATP displaced the binding of [³⁵S]ATPγS in tet− cell membranes with very high affinity, yielding pIC₅₀ values of 9.4±0.2 and 7.5±0.2, respectively. In tet+ cell membrane, displacement of [³⁵S]ATPγS by 2MeSATP and αβMeATP was of much lower affinity (pIC₅₀ values of 7.8 and 6.2, respectively). ATP, ADP and UTP showed similar displacement of [³⁵S]ATPγS binding in tet− and tet+ cell membranes. In other experiments, cytosolic Ca²⁺ was monitored using the fluorescent indicator, fluo-3. Increases in cytosolic Ca²⁺ were elicited by 100 nM αβMeATP in tet− cells while no increases in cytosolic Ca²⁺ were detected below 100 μM αβMeATP in either tet+ cells or untransfected cells. These calcium responses to αβMeATP had a pEC₅₀ of 6.7 and were transient, returning to baseline within 120 s. Suramin produced concentration-dependent, parallel, dextral shifts of E/[A] curves to αβMeATP yielding a pK_B of 5.6. PPADS produced non-parallel, dextral shifts of E/[A] curves to αβMeATP which were insurmountable. These results show for the first time, expression of a functional, homomeric recombinant rat P2X₃ receptor which is under regulated expression in a stably transfected mammalian cell line.

Our understanding of the actions of extracellular ATP in controlling kidney function via stimulation of P2 receptors is still at an early stage. Recently, several groups, including our own, have begun to address this subject: in this brief review, we discuss some of these effects and speculate on likely function of extracellular nucleotides in the kidney.

The endothelial cells relase both relaxing [nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), prostacyclin] and contracting factors [endoperoxides, thromboxane A₂, superoxide anions, endothelin-1 (ET)]. The production of ET is inhibited by NO. The latter also strongly opposes the direct effects of the former on vascular smooth muscle. With aging and vascular disease, the production of enothelial NO declines, and thus ET can be released, act and contribute to the symptoms.

We have previously shown that myoendothelial gap junctions are more prevalent in distal than in proximal arteries of the rat mesentery. In the present study we have investigated the role of gap junctions in the mechanism of action of endothelium-derived hyperpolarizing factor (EDHF) in these same vessels following relaxation with acetylcholine. Arteries were pre-constricted with phenylephrine and concentration response curves to acetylcholine were constructed in the presence of N^G-nitro-l-arginine methyl ester (l-NAME; 10⁻⁵ M) and indomethacin (10⁻⁵ M) to prevent effects due to the release of nitric oxide and prostacyclins. Nitric oxide was found to have only a small role in the relaxation of the proximal vessels and was not involved in the relaxations of the distal vessels. 18α-Glycyrrhetinic acid (10⁻⁵ M), a putative gap junction uncoupler, significantly reduced acetylcholine-induced relaxations by 50% in both proximal and distal vessels. Potassium channel antagonists, tetraethylammonium chloride (TEA; 10⁻³ M) and barium chloride (10⁻⁴ M), together abolished the dilatory response in the proximal mesenteric arteries, but did not completely block responses in the distal arteries. The data suggest that gap junctions contribute significantly to the acetylcholine-induced relaxation in both proximal and distal arteries of the rat mesentery. We hypothesize that the absence of a correlation between the role of gap junctions and the incidence of myoendothelial gap junctions in these same vessels is due to significant effects of the inhibitors on gap junctions located in the smooth muscle layers of the larger vessels.

1,4-Dihydropyridines are regarded as privileged structures for drug design, i.e. they tend to bind to a wide variety of receptor sites. We have shown that upon appropriate manipulation of the substituent groups on a 1,4-dihydropyridine template, high affinity and selectivity for the A₃ subtype of adenosine receptors (‘P1 receptors’) may be attained. In the present study we have begun to extend this approach to P2 receptors which are activated by ATP and other nucleotides. Nicardipine, a representative dihydropyridine, used otherwise as an L-type calcium channel blocker, was shown to be an antagonist at recombinant rat P2X₂ (IC₅₀=25 μM) and P2X₄ (IC₅₀ ∼220 μM) receptors expressed in Xenopus oocytes. Thus, this class of compounds represents a suitable lead for enhancement of affinity through chemical synthesis. In an attempt to modify the 1,4-dihydropyridine structure with a predicted P2 receptor recognition moiety, we have replaced one of the ester groups with a negatively charged phosphonate group. Several 4-phenyl-5-phosphonato-1,4-dihydropyridine derivatives, MRS 2154 (2,6-dimethyl), MRS 2155 (6-methyl-2-phenyl), and MRS 2156 (2-methyl-6-phenyl), were synthesized through three component condensation reactions. These derivatives were not pure antagonists of the effects of ATP at P2X₂ receptors, rather were either inactive (MRS 2156) or potentiated the effects of ATP in a concentration-dependent manner (MRS 2154 in the 0.3–10 μM range and MRS 2155 at >1 μM). Antagonism of the effects of ATP at P2X₂ receptor superimposed on the potentiation was also observed at >10 μM (MRS 2154) or 0.3–1 μM (MRS 2155). Thus, while a conventional dihydropyridine, nicardipine, was found to antagonize rat P2X₂ receptors ninefold more potently than P2X₄ receptors, the effects of novel, anionic 5-phosphonate analogues at the receptor were more complex.

Extracellular purine and pyrimidine nucleotides modulate cellular activity by acting at P2 receptors. The first receptor to be identified was the P₂-purinoceptor, which was characterised and named in 1978. In the 1980s this site was subdivided into P_2X and P_2Y purinoceptors on the basis of pharmacological criteria in functional studies on native receptors. Subsequently, a similar approach led to the characterisation of the P_2T, P_2Z, P_2U and P_2D purinoceptors. In the 1990s a molecular biological approach has led to the cloning and functional expression of at least 12 mammalian P2 receptor subtypes. The challenge now is to relate these recombinant receptors to native receptors present within a wide range of tissues.

Geoff Burnstock’s remarkable insight and tenacity has established the area of purinergic research as a bona fide target for drug discovery. While efforts in P1 receptor-based medicinal chemistry and biology efforts over the past 25 years have not reached the level of success that the pharmaceutical industry investment may have anticipated, the P2 area, with knowledge of the selective localization of members of the P2X and P2Y family members and data from transgenic knockouts, has identified several potential therapeutic areas of major promise including cystic fibrosis, chronic bronchitis, male contraception and neurodegeneration. In addition, interest in the potential of purinergic therapeutics has extended outside the major pharmaceutical companies to the ‘biotech industry’ resulting in an environment where the inherent risks of ‘first in field’ in a therapeutic area may be more appropriately nurtured.