Concetta Harakal

The effects of N-methyl-D-aspartate agonists and antagonists on isolated bovine cerebral arteries

This pharmacologic study examines the direct cerebrovascular effects of N-methyl-D-aspartate (NMDA) receptor agonists and antagonists to determine whether large cerebral arteries have NMDA receptors. Bovine middle cerebral arteries were cut into rings to measure isometric tension development in vitro. Two competitive agonists, L-glutamate and NMDA, each had negligible effects on ring tension in the absence of exogenous vasoconstrictors. L-glutamate (in high concentrations) produced direct relaxation of potassium (K+)-constricted arteries, but the relaxation was not selective for L-glutamate, D-glutamate, or mannitol. Relaxation with L-glutamate was abolished when it was isosmotically substituted in the K+-rich medium. NMDA (in the absence or presence of glycine) and two competitive antagonists, 2-amino-5-phosphopentanoic acid (AP5) and (+/-)-3-(s-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), each had little effect on the tone of arteries preconstricted with potassium or the stable thromboxane A2 analog U-46,619. Three noncompetitive antagonists (S(+)-ketamine, dizocilpine, and dextrorphan) and their stereoisomers (R(-)-ketamine, (-)MK-801, and levorphanol) each produced dose-dependent relaxation of K+- or U-46,619-constricted arteries; relaxation was not selective for the (+) or (-) stereoisomers. These results suggest that large cerebral arteries lack NMDA receptors mediating constriction or relaxation. All noncompetitive antagonists dilated cerebral arteries, but by mechanisms that were not stereospecific. (Anesth Analg 1996;82:264-8)

Ketamine directly dilates bovine cerebral arteries by acting as a calcium entry blocker.

This in vitro study was performed to determine the role of calcium in ketamine-induced cerebral vasodilation. Isolated bovine middle cerebral arteries were cut into rings to measure isometric tension development or into strips to measure radioactive 45Calcium (45Ca) uptake. Ketamine produced direct relaxation of arterial rings; the relaxation was attenuated in Ca(2+)-deficient media. Ketamine produced dose-related relaxation of arteries preconstricted with potassium, a stable thromboxane A2 analogue, or endothelin. Endothelial stripping with Triton X-100 had no effect on subsequent ketamine-induced relaxation. In Ca(2+)-deficient media containing potassium or the stable thromboxane A2 analogue, ketamine produced competitive inhibition of subsequent Ca(2+)-induced constriction. Ketamine blocked potassium- and thromboxane A2-stimulated 45Ca uptake in a dose-dependent manner, but had no effect on basal 45Ca uptake, the externally bound 45Ca content, or the volume of the 3H-sorbitol space. These results indicate that ketamine can directly dilate cerebral arteries by acting as a calcium channel antagonist; ketamine inhibits 45Ca uptake through both potential-operated (potassium) and receptor-operated (thromboxane A2) channels in cerebrovascular smooth muscle.

Effects of calcium antagonists on isolated bovine cerebral arteries: inhibition of constriction and calcium-45 uptake induced by potassium or serotonin.

The purpose of this study was to determine the mechanisms by which organic calcium channel blockers inhibit cerebral vasoconstriction. Isolated bovine middle cerebral arteries were cut into rings to measure contractility or into strips to measure radioactive calcium (45Ca) influx and efflux. Calcium channel blockers (10(-5) M verapamil or 3.3 X 10(-7) M nifedipine) and calcium-deficient solutions all produced near-maximal inhibition of both potassium- and serotonin-induced constriction. In calcium-deficient solutions containing potassium or serotonin, verapamil and nifedipine each blocked subsequent calcium-induced constriction in a competitive manner. Potassium and serotonin significantly increased 45Ca uptake into cerebral artery strips during 5 minutes of 45Ca loading; for potassium 45Ca uptake increased from 62 to 188 nmol/g, and for serotonin from 65 to 102 nmol/g. Verapamil or nifedipine had no effect on basal 45Ca uptake but significantly blocked the increase in 45Ca uptake induced by potassium or serotonin. Potassium, and to a lesser extent serotonin, each induced a brief increase in the rate of 45Ca efflux into calcium-deficient solutions. Verapamil or nifedipine had no effect on basal or potassium-stimulated 45Ca efflux. The results demonstrate that verapamil and nifedipine block 45Ca uptake through both potential-operated (potassium) and receptor-operated (serotonin) channels in bovine middle cerebral arteries.

N-methyl-D-aspartate (NMDA) antagonists--S(+)-ketamine, dextrorphan, and dextromethorphan--act as calcium antagonists on bovine cerebral arteries.

Ketamine, an intravenous anesthetic and a major drug of abuse, is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. Ketamines enantiomer, S(+)-ketamine, acts stereoselectively on neuronal NMDA receptors. The purpose of this in vitro study was to compare the direct effects of S(+)-ketamine, 2 other noncompetitive NMDA receptor antagonists (dextrorphan and dextromethorphan), and the calcium entry blocker nimodipine on the cerebral vasculature, using bovine middle cerebral arteries as an experimental model. Arterial rings were mounted in isolated tissue chambers equipped with isometric tension transducers to obtain pharmacologic dose-response curves. In the absence of exogenous vasoconstrictors, the NMDA antagonists or nimodipine had negligible effects on cerebral arterial tone. When rings were preconstricted with either potassium or the stable thromboxane A2 mimetic U46619, the NMDA antagonists and nimodipine each produced dose-dependent relaxation. Prior endothelial stripping had no effect on subsequent drug-induced relaxation of K+-constricted rings. In Ca2+-deficient media containing either potassium or U46619, the NMDA antagonists and nimodipine each produced competitive inhibition of subsequent Ca2+induced constriction. In additional experiments, arterial strips were mounted in isolated tissue chambers to directly measure calcium uptake, using 45calcium (45Ca) as a radioactive tracer. The NMDA antagonists and nimodipine each blocked potassium-stimulated or U46619-stimulated 45Ca uptake into arterial strips. These results indicate that S(+)-ketamine, dextrorphan, and dextromethorphan, like nimodipine, directly dilate cerebral arteries by acting as calcium antagonists; they all inhibit 45Ca uptake through both potential-operated (potassium) and receptor-operated (U46619) channels in cerebrovascular smooth muscle.

Gatecholamine Content and Response to Norepinephrine of Middle Cerebral Artery

Summary The norepinephrine (NE) and epinephrine (E) contents of bovine middle cerebral (MCA) and lingual (LA) arteries were determined fluorometrically. Although the amount of E was almost identical in the 2 vessels, the MCA contained almost four times as much NE as the LA. Cumulative NE dose-response curves revealed a much greater reactivity of the LA than the MCA. An inverse relationship is demonstrated between NE content and vascular reactivity to this agent, and a similar relationship is suggested between NE content and the degree of functional innervation in some vascular beds. The technical assistance of Mrs. Olha Holo-wecky is gratefully acknowledged.

Relaxation methods for the determination of drug receptor affinities.

According to the occupation theory of drug receptor interaction, the response is a functionf of the number of receptors occupied by drug molecules. Considerable controversy exists regarding assumptions about this function. Without knowledge of the nature of the function, it is not possible to determine directly the rate constants, and hence the affinity constant, in the reaction between the receptor and an agonist drug. Instead, indirect determinations involving the use of antagonists have been employed, limiting the determination of affinity to those agents for which specific antagonists exist. The present paper discusses a method for the direct determination of affinity of an agonist drug. It is a “relaxation method,” i.e., the equilibrium is perturbed and the kinetics of the restoration process are studied. Assuming only thatf is non-decreasing and approximately linear over a limited domain of concentrations, it is shown that the change in response obeys first order kinetics, permitting a determination of the rate constants from the time course of the restoration process.

Mediation of bradykinin‐induced contraction in canine veins via thromboxane/prostaglandin endoperoxide receptor activation

1 Canine jugular and femoral veins were studied to determine the possible importance of thromboxane (TXA2) and prostaglandin endoperoxides (prostaglandin H2, PGH2) in mediating bradykinin(BK)‐induced contraction. 2 Isolated vein rings incubated in modified Krebs solution contracted to TXA2/PGH2 analogs SQ26655 and U44069 with potency of contraction exceeding that for BK. The potency ranking for both veins was SQ26655 > U44069 > BK > PGF2α > TXB2 > PGD2. 3 The cyclo‐oxygenase inhibitors indomethacin (3 × 10−7 m) and flufenamic acid (10−5 m) reduced BK contractions without affecting those induced by noradrenaline (NA). 4 TXA2/PGH2 receptor antagonists SQ29548 (10−8 m) and BM13177 (10−6 m) strongly inhibited BK‐induced tension. The action of antagonists was reversible with negligible influence on NA‐elicited contraction. Selective removal of endothelium had no effect on BK‐induced contraction or the action of the antagonists. 5 The thromboxane synthase inhibitors dazoxiben (10−4 m) and CGS 12970 (10−5 m) had no significant inhibitory effect on BK‐induced tension. 6 These results suggest that in canine jugular and femoral vein, the action of BK is largely dependent upon stimulation of the cyclo‐oxygenase pathway to produce PGH2 and possibly TXA2, which can activate a smooth muscle TXA2/PGH2 receptor to elicit vasoconstriction.

Calcium fractions in aortic segments from hypertensive rabbits.

The rate constants (-k) of Ca2+ from cellular compartments of intact smooth muscle cells of thoracic aorta of normotensive, renal hypertensive, and cadmium hypertensive rabbits were studied by 45Ca efflux and by employing norepinephrine (NE, 5.9 × 10-7 M). Tissues from hypertensive animals showed an increased rate of release of 45Ca over a 5-h unloading period when compared to normotensive controls. Three exchangeable compartments were found to contribute to the total kinetic efflux curve for all groups. The Ca2+ fraction in compartments I and II showed an increased rate of Ca2+ release for the hypertensive subgroups. Although the rates were slowed for tissues treated with NE prior to efflux, the rates of the hypertensive subgroups were increased compared to the normotensive group. Pretreatment of aortic segments with NE caused an overall increase in 45Ca activity of the incubation medium and may explain the slowed rates attained in the efflux analyses. Influx studies showed that tissues from hypertensive animals had a greater uptake of 45Ca than did the normotensive controls. Such data may represent a larger exchangeable component in aortae from the hypertensive subgroups. These findings indicate differences in the rates of Ca2+ release from intact smooth muscle cells of hypertensive rabbits.

An index for comparing the inhibitory action of vasodilators

An index for comparing the inhibitory effects of vasodilators was developed to gain insight into their mechanism of action on vascular smooth muscle. Rat aortic strips were bathed in Krebs bicarbonate solution and were initially contracted to a stable tension by either phenylephrine or barium chloride. A vasodilator was then added and the remaining tension was noted; this was repeated for cumulative concentrations of vasodilator. At each concentration of vasodilator, the percent reduction in phenylephrine-induced tension (Phe) was compared to the percent reduction in barium-induced tension (Ba) and was expressed as a ratio (Phe/Ba). This ratio clearly separated verapamil and nifedipine (ratio less than 1), which block calcium influx, from papaverine (ratio = 1) which promotes calcium sequestration regardless of the source of calcium, and from dantrolene (ratio greater than 1) which interferes with intracellular calcium mobilization. This index provides a method for comparing the action of those agents presently classified as non-receptor specific vasodilators which act directly on vascular smooth muscle.

Cyclic nucleotide changes in aortic segments derived from hypertensive rabbits.

Mean blood pressure increase was produced by: (1) renal constriction plus contralateral nephrectomy, and (2) cadmium acetate ingestion. Blood pressure was measured directly via the middle ear artery. Cyclic nucleotide levels were determined by 125I-radioimmunoassay. Levels of cAMP were reduced in both hypertensive subgroups when compared to controls. The altered cAMP levels suggest a relationship of this nucleotide to the maintenance of increased blood pressure.