Hall Downes

Forebrain Ischemia Increases Glut1 Protein in Brain Microvessels and Parenchyma

Glucose transport into nonneuronal brain cells uses differently glycosylated forms of the glucose transport protein, GLUT1. Microvascular GLUT1 is readily seen on immunocytochemistry, although its parenchymal localization has been difficult. Following ischemia, GLUT1 mRNA increases, but whether GLUT1 protein also changes is uncertain. Therefore, we examined the immunocytochemical distribution of GLUT1 in normal rat brain and after transient global forebrain ischemia. A novel immunocytochemical finding was peptide-inhibitable GLUT1 immunoreactive staining in parenchyma as well as in cerebral microvessels. In nonischemic rats, parenchymal GLUT1 staining co-localizes with glial fibrillary acidic protein (GFAP) in perivascular foot processes of astrocytes. By 24 h after ischemia, both microvascular and nonmicrovascular GLUT1 immunoreactivity increased widely, persisting at 4 days postischemia. Vascularity within sections of brain similarly increased after ischemia. Increased parenchymal GLUT1 expression was paralleled by staining for GFAP, suggesting that nonvascular GLUT1 overexpression may occur in reactive astrocytes. A final observation was a rapid expression of inducible heat shock protein (HSP)70 in hippocampus and cortex by 24 h after ischemia. We conclude that GLUT1 is normally immunocytochemically detectable in cerebral microvessels and parenchyma and that parenchymal expression occurs in some astroglia. After global cerebral ischemia, GLUT1 overexpression occurs rapidly and widely in microvessels and parenchyma; its overexpression may be related to an immediate early-gene form of response to cellular stress.

Binding of thiopental in neonatal serum.

Protein binding of thiopental was studied in 21 samples of neonatal serum (from placental blood) and compared with protein binding in ten healthy volunteers. These infants ranged between 32 and 43 weeks of gestational age (mean, 37.7 weeks) and the adult age range was from 27 to 54 years (mean, 35.4 years). Because the unbound fraction of the drug is responsible for its pharmacologic effect, a marked difference in the protein binding between neonates and adults may be relevant to the clinician. Blood obtained from freshly delivered placentas or from adult volunteers was allowed to clot and the serum separated from the sample. A portion of the serum was sent for protein and bilirubin analysis and the remainder retained for study. This latter serum was combined with four concentrations of thiopental. These specimens were then ultrafiltered and the amount of thiopental in the ultrafiltrate (unbound) compared with the prefiltered amount (total), as measured by reverse-phase high-performance liquid chromatography. The binding studies were repeated at pH 7.2, 7.4, and 7.6 in both the adult and neonatal serum. Total protein and albumin are significantly less in neonatal serum, whereas bilirubin (total and direct) is significantly higher in neonatal serum than in adult serum (P less than 0.01). Neonatal serum was associated with significantly more unbound thiopental than adult serum at all levels of pH studied (P less than 0.005). Increasing the pH resulted in less free drug in both groups, but this reached statistical significance only in the adult group (P less than 0.025). Drug concentration had no effect on binding in the range examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Airway responses to methacholine and histamine in basenji greyhounds and other purebred dogs

To investigate whether airway hyperresponsiveness is a general characteristic of either the basenji or the greyhound ancestry of the basenji-greyhound (BG) dog and to determine whether airway hyperresponsiveness of the BG dogs in specific for methacholine, we compared pulmonary responsiveness to increasing doses of aerosols of histamine and methacholine in 17 BG dogs, 5 unrelated purebred basenjis and 5 unrelated greyhounds. BG dogs were hyperresponsive to both methacholine and histamine compared to basenjis and greyhounds. There was a significant correlation between histamine compared to basenjis and greyhounds. There was a significant correlation between histamine and methacholine responsiveness (r = 0.70, P less than 0.0001) in the dog population. We conclude that airway hyperresponsiveness characteristic of the BG dog is not a general characteristic of the basenji or greyhound breed and occurs to histamine as well as to methacholine.

Distinctive pharmacological profile of a nonadrenergic inhibitory system in bullfrog lung

1 Bullfrog hemilungs, pretreated with atropine, are markedly relaxed on addition of carbachol. Since the relaxant effect is inhibited by tetrodotoxin or hexamethonium, it is neurally mediated and involves stimulation of nicotinic receptors with release of an unknown inhibitory transmitter. 2 Carbachol‐induced relaxation is nonadrenergic since: (a) it considerably exceeds the maximal effects of isoprenaline or the effect of 10−3m adrenaline or noradrenaline; (b) it elicits marked further relaxation in preparations already relaxed by high concentrations of catecholamines; (c) it is not attenuated by low concentrations of propranolol (10−6 and 3 × 10−6m) that competitively antagonize isoprenaline‐induced relaxation. 3 Carbachol‐induced relaxation has multiple distinguishing characteristics, which serve as a fingerprint for the unknown inhibitory transmitter. These include an exceptionally rapid onset of action, a ceiling effect at 50% of maximal relaxation, and minimal retardation by concentrations of procaine that block or markedly retard relaxant responses to all other agonists. 4 This distinctive pharmacological profile cannot be reproduced by addition of exogenous catecholamines, 5‐hydroxytryptamine, adenosine triphosphate (ATP) or adenosine, or by addition of ATP or adenosine following pretreatment with indomethacin. Furthermore, addition of carbachol to preparations previously relaxed with 10−3m concentrations of these agents produced marked, additional relaxation. 5 Maximally effective concentrations of vasoactive intestinal peptide produced a barely detectable relaxant response equivalent to 8% of maximal relaxation. The response was totally prevented by pretreatment with procaine. 6 Carbachol‐induced relaxation was not impaired by pretreatment with 10−4m indomethacin. 7 Carbachol‐induced relaxation of bullfrog lung therefore involves a postganglionic inhibitory transmitter that in nonadrenergic, non‐5‐hydroxytryptaminergic, and nonpurinergic, and whose effects are not dependent on prostaglandin synthesis. Although a peptide may function as the inhibitory transmitter, it is not vasoactive intestinal peptide.

Muscle weakness during tricaine anesthesia

An isolated preparation of tadpole tail muscle was used to assess the peripheral effects of tricaine (3-aminobenzoic acid ethyl ester) at anesthetic concentrations and under physiological conditions. The drug effect on the electrically-evoked twitch was tested using short-pulse durations that elicited synaptically mediated effects or longer-duration pulses that stimulated the muscle directly. Tricaine reduced both types of response anesthetic and even subanesthetic concentrations. At steady state concentrations that produced surgical anesthesia in vivo, tricaine reduced the directly evoked response by about half. It is concluded that tricaine anesthesia has a pronounced peripheral effect on neuromuscular function and that direct effect(s) on muscle are a major component.

Metamorphosis and the steady state anesthetic concentrations of tricaine, benzocaine and ethanol

Abstract In the classical tadpole assay employed for Meyer-Overton type correlations, tricaine and benzocaine are 7–8 times more potent than n -alkanols of equivalent lipid solubility. Median anesthetic concentrations for loss of the righting reflex, AC 50 (RR)s, were 0.165 and 0.103 mM for tricaine and benzocaine, and log P s (octanol: water) were 1.81 and 1.98, respectively. Tadpoles receiving a half AC 50 (RR) each of tricaine and ethanol showed less than additive effects, suggesting a substantial difference in their mechanism(s) of action. AC 50 (RR)s for both tricaine and benzocaine increased two fold coincident with metamorphosis, reflecting a change from an equilibrium to a nonequilibrium steady state.

Retention of nociceptor responses during deep barbiturate anesthesia in frogs

Bullfrogs (Rana catesbeiana) anesthetized with a large dose of thiopental (42.8 mg/kg) retained movement responses to nociceptor stimuli despite an average plasma drug level of 51 mg/l, of which 63% was bound to plasma proteins. This concentration, when corrected to include only unbound and uncharged drug, was 2-fold greater than those reported to abolish nociceptor response (NR) during surgical anesthesia in man. The median anesthetic dose (AD50) for loss of the righting reflex was 11.2 mg/kg by s.c. injection into the abdominal lymph sac; however, at 54.0 mg/kg, all frogs retained NRs, although otherwise deeply anesthetized. The ratio of NR-blocking dose to light AD was thus > 4.8, as compared to < 2 in mammalian studies. Whole body levels of thiopental determined at 3 h after intralymphatic injection showed that about half the injected drug had been eliminated by this time and that termination of anesthesia was chiefly due to drug elimination. Even though the pharmacokinetics of thiopental appears to differ markedly in frogs and men, the poor analgesia seen in the present study frequently has been reported during clinical barbiturate anesthesia. Since this deficiency is much more pronounced in the bullfrog than in man, its neurophysiological basis might profitably be studied using the bullfrog as a model; however, the high mortality associated with deep thiopental anesthesia in the frog should preclude its use as a practical anesthetic in amphibia.

Mechanisms of intrinsic tone in bullfrog lung: relaxant effects of indomethacin, ouabain and potassium.

1 Bullfrog hemilungs showed minimal relaxation (9 ± 2% of the maximal relaxant effect of theophylline, Imax) after a 16 h incubation in 10−5 M indomethacin, indicating that prostaglandin synthesis plays little or no role in the high intrinsic tone characteristic of this preparation. 2 A higher concentration of indomethacin (10−4 M) produced greater relaxation (23 ± 3% of Imax), but also markedly potentiated isoprenaline‐induced relaxation. The interaction with isoprenaline was similar to that previously found for papaverine, a phosphodiesterase inhibitor. 3 Ouabain (10−5 and 10−4 M) produced an initial contraction followed by marked relaxation (50% of Imax), indicating that a ouabain‐sensitive mechanism is of major importance in the maintenance of intrinsic tone. 4 Ouabain‐treated hemilungs showed (a) reversal (relaxation) of the normal contractile response to 26 mM potassium and (b) marked impairment of the contractile response to calcium in calcium‐depleted preparations. These effects suggest that ouabain‐induced relaxation reflects a drug action on calcium movements. 5 The marked relaxation (30 to 40% of Imax) produced by 26 mM potassium in ouabain‐treated hemilungs is of particular interest in that it indicates a mechanism of potassium‐induced relaxation distinct from stimulation of sodium‐potassium ATPase.

An anticholinergic effect of hexylcaine on airway smooth muscle.

Hexylcaine, an eater-type local anesthetic, was studied in guinea pig tracheal chains for its comparative effects on intrinsic tone and on responses to carbamylcholine, histamine and isoproterenol. Drug effects were recorded isotonically at a bath (PH of 7.5 (CO2, 5 per cent, and 25 mM bicarbonate) or (PH 6.75 (CO2, 5 per cent, and 5.75 mM bicarbonate), corresponding, respectively, to nonionized drug concentrations of 5.68 and 1.05 per cent of total hexylcaine. Low concentrations (10 to 3±10-4M) of hexylcaine produced an apparently competitive antagonism (P A2=4.95) of carbamylcholine. The extents of antagonism were not significantly different at the two values of experimental(PH, indicating that nonionized drug was not essential to the anticholinergic effect. In contrast, the concentration necessary for relaxation of intrinsic tone changed with (PH(10-3M at (PH 7.5; 3±10-3M at (PH 6.75), indicating that nonionized drug was essential, for access or for action, to the nonspecific relaxant effects of high drug concentrations. Hexylcaine, 3±10-4M(PH 7.5), increased the concentrations of carbamylcholine and histamine needed to produce a half-maximal response by 20.9 times and 3.6 times, respectively, and had no effect on responses to isoproterenol. The authors conclude that hexylcaine has selective and apparently competitive anticholinergic effects that are manifest at clinically relevant concentrations and are mechanistically distinct from the general depressant effects of higher concentrations.

Effect of stimulation of non-adrenergic inhibitory nerves on cyclic nucleotide levels in bullfrog lung

Carbachol in the presence of atropine and propranolol was employed to stimulate a non-adrenergic neural inhibitory system in the hemilung of the bullfrog (Rana catesbeiana). Tissue levels of cGMP were elevated 95% by carbachol whilst cAMP levels were unchanged. The phosphodiesterase inhibitor papaverine did not affect either cAMP or cGMP levels, but did selectively increase the carbachol-induced increase in lung cGMP to 220% of control levels. Papaverine did not potentiate the relaxant effects of carbachol. The results suggest that cyclic nucleotides may not be directly involved in the relaxation produced by stimulation of the non-adrenergic neural inhibitory system in this preparation.