Enhancer substances: selegiline and R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane [(-)-BPAP] enhance the neurotrophic factor synthesis on cultured mouse astrocytes

R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane [(-)-BPAP] is a potent "catecholaminergic and serotonergic activity enhancer (CAE/SAE)", which enhances the impulse-evoked catecholamines and serotonin release, e.g. (-)-BPAP enhances in vitro norepinephrine efflux from the slices of locus coeruleus in a bipolar manner with the two effective ranges of low (fM-pM level) and high (nM-microM level) concentrations. Here, the effects of (-)-BPAP and selegiline on the cultured mouse astrocytes were studied. The protein levels of the neurotrophic factors (NGF, BDNF and GDNF) in the conditioned medium of cultured astrocytes were determined by using ELISA. In the cultured astrocytes incubated for 24 h with selegiline, the synthesis of NGF and BDNF was significantly enhanced in the concentration dependent manner, with minimum effective concentrations of 4 x 10(-4) and 5 x 10(-4) M, respectively. (-)-BPAP also enhanced the NGF, BDNF and GDNF synthesis, with minimum effective concentrations of 5 x 10(-5), 1 x 10(-5), and 1 x 10(-6) M, respectively. Although the effects of (-)-BPAP on the NGF synthesis was tested in the range of 1 x 10(-15)-5 x 10(-4) M, the concentration response curve of (-)-BPAP was a single bell shape with the peak effect at 1 x 10(-4) M, and did not show any effects in low concentrations such as fM-pM level. Each concentration response curve of (-)-BPAP on BDNF and GDNF synthesis was a single bell shape with peak effects at 1 x 10(-3) M and 1 x 10(-4) M, respectively.     

Effects of amiridin and tacrine, drugs effective in Alzheimer's disease, on the activity of monoamine oxidase A and B]

In vitro comparative studies of effects of amiridin (9-amino-2, 3, 5, 6, 7, 8-hexahydro-1H-cyclopentane (b) choline monohydrate hydrochloride) and tacrine physostigmine and piracetam on monoamine oxidase A (MAO-A) and B (MAO-B) activity in the rat brain were carried out. Piracetam (1 x 10(-4)-1 x 10(-3) M) dose-dependently increased MAO-A and MAO-B activity. At all concentrations used (1 x 10(-7)-5 x 10(-4) M) physostigmine had no effect on MAO-A and MAO-B activity. Amiridin was found to inhibit MAO-B activity at 5 x 10(-4) M concentration only. Tacrine inhibited MAO-A activity at 5 x 10(-4) M concentration. The therapeutical effects of amiridin and tacrine in treatment of Alzheimer disease were not related to their action on MAO-A and -B activity.     

Determination of agmatine in biological samples by capillary electrophoresis with chemiluminescence detection

A fast and simple method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of agmatine, a recently identified neurotransmitter/modulator. The CE run time was approximately 2 min for each sample injected. CL detection employed a lab-built reaction flow cell and a photon counter. The CL reagents used were luminol and NaBrO. The optimized conditions for the CL detection were 5 x 10(-4)M luminol added to the CE running buffer and 5.0 x 10(-4)M NaBrO in 100 mM NaCO3-NaOH buffer solution at pH 12.5 introduced post column. Detection limit for agmatine was 4.3 x 10(-6)M (S/N=3). The precision (R.S.D.) on peak height (at 1 x 10(-5)M agmatine) and migration time were 3.7 and 2.5%, respectively. The present CE-CL method was evaluated with the determination of agmatine in tissue samples taken from rat brain, and rat and monkey stomachs. Samples were directly injected into the CE-CL system after the removal of proteins. A higher level of agmatine was detected in the stomach samples. Agmatine concentrations in the tissue samples taken from rat and monkey stomachs were similar at approximately 1950 ng/g wet tissue.     

Effects of norepinephrine on lung liquid production by in vitro lungs from fetal guinea pigs

Lungs from near-term fetal guinea pigs (61 +/- 2 days of gestation) were supported in vitro for 3 h; lung liquid production was monitored by a dye dilution method. Untreated control preparations produced fluid at 1.38 +/- 0.30 mL x kg(-1) body weight x h(-1), with no significant change (ANOVA; regression analysis); those given 1.24 x 10(-9) or 1.24 x 10(-8) M norepinephrine during the middle hour showed no significant change, but those given concentrations between 5.24 x 10(-8) and 1.24 x 10(-5) M all showed significant reductions or fluid reabsorption (based on 42 fetuses). The responses showed a linear relationship with the log concentration (r = 0.97). They appeared to involve alpha-adrenoreceptors, since responses to 10(-7) M norepinephrine were unaffected by 10(-6) M propranolol, but those to 10(-7) and 1.24 x 10(-6) M norepinephrine were abolished by 10(-6) and 1.78 x 10(-5) M phentolamine, respectively (based on 48 fetuses). Activation was through alpha2-adrenoreceptors, since responses to 10(-7) and 10(-5) M norepinephrine were abolished by 10(-4) M yohimbine, but not by 10(-5) M prazosin (based on 60 fetuses). The results show that norepinephrine is able to reduce lung liquid production when at plasma levels present at birth, and that it can produce reabsorption; unlike epinephrine, there was no reduction in responses at high concentrations. This work reintroduces a neglected factor, norepinephrine, into possible controls of lung liquid reabsorption, and opens up the potential for neural controls.     

Effect of chlorpromazine, imipramine and lithium on MAO-A and MAO-B activity in rat brain mitochondria

Drugs with efficacy in psychiatric disorders affect the function of central neurotransmitter amines, which are inactivated primarily by monoamine oxidase (MAO). Effect of these drugs on the two types of MAO (MAO-A and MAO-B) has been studied in rat brain. The result showed that chlorpromazine (CPZ) and imipramine (IMI) at concentrations of 1x10(-2), 5x10(-3) and 2.5x10(-3) M inhibited rat brain mitochondrial MAO-A activity in vitro by 82, 50, 39 and 86, 74, 38 %, respectively. CPZ at concentrations of 5x10(-3), 2.5x10(-3), 1x10(-3) M inhibited rat brain mitochondrial MAO-B activity in vitro by 83, 55, 39 %, respectively, while IMI at concentrations of 5x10(-4), 2.5x10(-4), 1x10(-4) M inhibited the in vitro enzyme activity by 43, 35, 21 %, respectively. Lithium at concentration of 5x10(-3) M could not either inhibit MAO-A or MAO-B in the mitochondrial fraction of rat brain.     

Early adrenal infection by herpes simplex virus type-1 (Miyama + GC strain): special reference to inoculation dose and spread from the adrenal to the central nervous system

Male C3H/HeN mice, aged 5 weeks, were inoculated intraperitoneally (i.p.) with different doses (1 x 10(3), 1 x 10(5), 5 x 10(5), 1 x 10(6) pfu) of the herpes simplex virus type-1 (HSV-1) (Miyama + GC strain). The LD50 of this virus was 10(2) pfu (i.p.) per mouse. All the mice in each group died 12 days after inoculation. Adrenal necrosis was found to be dose-dependent, the threshold dose being 5 x 10(5) pfu. In addition, encephalitis and inflammatory cell infiltration in abdominal ganglia appeared in 3-4 days after inoculation. By the plaque method, HSV-1 was detected first in the adrenal glands, then in neurons in the spinal cord and the brain. These findings suggest that in mice inoculated with doses of virus sufficient to infect the adrenal gland, HSV-1 spreads to the central nervous system through peripheral nerves after replication in the adrenal.     

Prostaglandin F2 stimulates the generation of lipoxygenase products in guinea pig isolated trachea

The generation of lipoxygenase products on the contraction elicited by prostaglandin (PG) F2 alpha was investigated in the guinea-pig isolated trachea. Indomethacin (5 x 10(-6) M) inhibited the response at low concentrations of PGF2 alpha while enhanced the response at higher concentrations of PGF2 alpha. Phenidone (10(-4) M) and nordihydroguaiaretic acid (NDGA, 3 x 10(-5) M) appeared to inhibit the PGF2 alpha response. The PGF2 alpha response augmented by indomethacin was dose-dependently inhibited by NDGA and a leukotriene (LT) antagonist, FPL55712. NDGA had no effect on the contraction elicited by histamine but markedly inhibited the contraction elicited by LTD4. The inhibition by NDGA of the LTD4-induced contraction was abolished in the presence of indomethacin (5 x 10(-6) M). FPL55712 inhibited the LTD4-induced contraction but the extent of the antagonism was not changed by indomethacin. In the presence of indomethacin PGF2 alpha (10(-8) M) did not affect the LTD4 (3 x 10(-9) M) response but significantly enhanced the arachidonic acid (AA, 6.6 x 10(-5) M)-induced contraction. FPL55712 (3 x 10(-6) M) completely inhibited the AA response augmented by PGF2 alpha. These results suggest that lipoxygenase-mediated LT-like substances are released in the response at higher concentrations of PGF2 alpha on the guinea-pig isolated trachea, and the mode of action of PGF2 alpha is different from those of histamine and LTD4.     

The effects of drug complexation on the stability and conformation of human serum albumin

We report different analytical methods used to study the effects of 3\'-azido-3\'-deoxythymidine, aspirin, taxol, cisplatin, atrazine, 2,4-dichlorophenoxyacetic, biogenic polyamines, chlorophyll, chlorophyllin, poly(ethylene glycol), vanadyl cation, vanadate anion, cobalt-hexamine cation, and As2O3, on the stability and secondary structure of human serum albumin (HSA) in aqueous solution, using capillary electrophoresis, Fourier transform infrared, ultraviolet visible, and circular dichroism (CD) spectroscopic methods. The concentrations of HSA used were 4% to 2% or 0.6 to 0.3 mM, while different ligand concentrations were 1 microM to 1 mM. Structural data showed drugs are mostly located along the polypeptide chains with both specific and nonspecific interactions. The stability of drug-protein complexes were in the order K(VO(2+)) 1.2 x 10(8) M(-1) > K(AZT) 1.9 x 10(6) M(-)1 > K(PEG) 4.1 x 10(5) M(-1) > K(atrazine) 3.5 x 10(4) M(-1) > K(chlorophyll) 2.9 x 10(4) M(-1) > K2,4-D 2.5 x 10(4) M-1 > K(spermine) 1.7 x 10(4) M(-1) > K(taxol) 1.43 x 10(4) M(-1) > K(Co(3+)) > 1.1 x 10(4) M(-1) > K(aspirin) 1.04 x 10(4)i(-1) > K(chlorophyllin) 7.0 x 10(3) M(-1) > K(VO(3)(-)) 6.0 x 103 M(-1) > K(spermidine) 5.4 x 10(3) M(-1) > K(putrescine) 3.9 x 10(3) M(-1) > K(As(2)O(3)) 2.2 x 10(3) M(-1)> K(cisplatin) 1.2 x 10(2) M(-1). The protein conformation was altered (infrared and CD results) with major reduction of alpha-helix from 60 to 55% (free HSA) to 49 to 40% and increase of beta-structure from 22 to 15% (free HSA) to 33 to 23% in the drug-protein complexes. The alterations of protein secondary structure are attributed to a partial unfolding of HSA on drug complexation.     

Specificity and affinity of binding of herpes simplex virus type 2 glycoprotein B to glycosaminoglycans.

Herpes simplex virus type 2 (HSV-2) interacts with cell surface glycosaminoglycans during virus attachment. Glycoprotein B of HSV-2 can potentially mediate the interaction between the virion and cell surface glycosaminoglycans. To determine the specificity, kinetics, and affinity of these interactions, we used plasmon resonance-based biosensor technology to measure HSV-2 glycoprotein binding to glycosaminoglycans in real time. The recombinant soluble ectodomain of HSV-2 gB (gB2) but not the soluble ectodomain of HSV-2 gD bound readily to biosensor surfaces coated with heparin. The affinity constants (Kds) were determined for gB2 (Kd = 7.7 x 10(-7) M) and for gB2 deltaTM (Kd = 9.9 x 10(-7) M), a recombinant soluble form of HSV-2 gB in which only its transmembrane domain has been deleted. gB2 binding to the heparin surface was competitively inhibited by low concentrations of heparin (50% effective dose [ED50] = 0.08 microg/ml). Heparan sulfate and dermatan sulfate glycosaminoglycans have each been suggested as cell surface receptors for HSV. Our biosensor analyses showed that both heparan sulfate and dermatan sulfate inhibited gB2 binding (ED50 = 1 to 5 microg/ml), indicating that gB2 interacts with both heparin-like and dermatan sulfate glycosaminoglycans. Chondroitin sulfate A, in contrast, inhibited gB2 binding to heparin only at high levels (ED50 = 65 microg/ml). The affinity and specificity of gB2 binding to glycosaminoglycans demonstrated in these studies support its role in the initial binding of HSV-2 to cells bearing heparan sulfate or dermatan sulfate glycosaminoglycans.     

Involvement of intracellular signaling factors in the serum-enhanced Ca2+-binding protein regucalcin mRNA expression in the cloned rat hepatoma cells (H4-II-E).

The involvement of signaling factors, which are related to serum component, on the regucalcin mRNA expression in the cloned rat hepatoma cells (H4-II-E) was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open reading frame). H4-II-E cells were cultured for 2 or 6 h in a medium containing various reagents in the presence of serum (10% fetal bovine serum) after the subconfluent with 3-day-culture. The regucalcin mRNA expression was significantly increased by serum addition. This increase was clearly inhibited by the presence of EGTA (10(-3) M), A23187 (10(-6) M), trifluoperazine (10(-5) M), staurosporine (10(-7) M), or genistein (10(-5) M) with 6-h-culture, although the beta-actin mRNA expression was not altered by the reagents. Meanwhile, the regucalcin mRNA expression was significantly stimulated by the addition of Bay K 8644 (2.5 x 10(-6) M) in the presence of serum. This effect was also seen in the presence of genistein (10(-5) M). The present study suggests that the regucalcin mRNA expression is mediated through signaling pathways which are partly involved in Ca2+-dependent protein kinases and tyrosine kinase in H4-II-E hepatoma cells.     

Soluble forms of herpes simplex virus glycoprotein D bind to a limited number of cell surface receptors and inhibit virus entry into cells.

Herpes simplex virus type 1 (HSV-1) and HSV-2 plaque production was inhibited by treating cells with soluble forms of HSV-1 glycoprotein D (gD-1t) and HSV-2 glycoprotein D (gD-2t). Both glycoproteins inhibited entry of HSV-1 and HSV-2 without affecting virus adsorption. In contrast, a soluble form of HSV-2 glycoprotein B had no effect on virus entry into cells. Specific binding of gD-1t and gD-2t to cells was saturable, and approximately 4 x 10(5) to 5 x 10(5) molecules bound per cell. Binding of gD-1t was markedly reduced by treating cells with certain proteases but was unaffected when cell surface heparan sulfate glycosaminoglycans were enzymatically removed or when the binding was carried out in the presence of heparin. Together, these results suggest that gD binds to a limited set of cell surface receptors which may be proteins and that these interactions are essential for subsequent virus entry into cells. However, binding of gD to its receptors is not required for the initial adsorption of virus to the cell surface, which involves more numerous sites (probably including heparan sulfate) than those which mediate gD binding.     

Inhibition of AChE by malathion and some structurally similar compounds

Inhibition of bovine erythrocyte acetylcholinesterase (free and immobilized on controlled pore glass) by separate and simultaneous exposure to malathion and malathion transformation products which are generally formed during storage or through natural or photochemical degradation was investigated. Increasing concentrations of malathion, its oxidation product malaoxon, and its isomerisation product isomalathion inhibited free and immobilized AChE in a concentration-dependent manner. KI, the dissociation constant for the initial reversible enzyme inhibitor-complex, and k3, the first order rate constant for the conversion of the reversible complex into the irreversibly inhibited enzyme, were determined from the progressive development of inhibition produced by reaction of native AChE with malathion, malaoxon and isomalathion. KI values of 1.3 x 10(-4) M(-1), 5.6 x 10(-6) M(-1) and 7.2 x 10(-6)M(-1) were obtained for malathion, malaoxon and isomalathion, respectively. The IC50 values for free/immobilized AChE, (3.7 +/- 0.2) x 10(-4) M/(1.6 +/-0.1) x 10(-4), (2.4 +/- 0.3) x 10(-6)/(3.4 +/- 0.1) x 10(-6)M and (3.2 +/- 0.3) x 10(-6) M/(2.7 +/- 0.2) x 10(-6) M, were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl ester and O,O-dimethyl thiophosphate, did not noticeably affect enzymatic activity, while diethyl maleate inhibited AChE activity at concentrations > 10mM. Inhibition of acetylcholinesterase increased with the time of exposure to malathion and its inhibiting by-products within the interval from 0 to 5 minutes. Through simultaneous exposure of the enzyme to malaoxon and isomalathion, an additive effect was achieved for lower concentrations of the inhibitors (in the presence of malaoxon/isomalathion at concentrations 2 x 10(-7) M/2 x 10(-7) M, 2 x 10(-7) M/3 x 10(-7)M and 2 x 10(-7) M/4.5 x 109-7) M), while an antagonistic effect was obtained for all higher concentrations of inhibitors. The presence of a non-inhibitory degradation product (phosphorodithioic O,O,S-trimethyl ester) did not affect the inhibition efficiencies of the malathion by-products, malaoxon and isomalathion.     

Properties of mannosyl- and N-acetylglucosamine-1-phosphate transferases towards dolichol phosphate in liver microsomes from pig embryos

1. The activity of mannosyl- and N-acetylglucosamine-1-phosphate transferases in microsomes from pig embryonic liver was linear to 1 min of incubation at 37 degrees C. 2. The activity of both enzymes was higher in the presence of Mg2+ as compared to Mn2+. A maximal stimulatory effect of Mn2+ was obtained at 2 mM concentration and greater concentrations of it inhibited the activities of both enzymes. 3. The activity of mannosyl transferase was found to be highest after treatment of microsomes with Nonidet P-40 while the activity of N-acetylglucosamine-1-phosphate transferase was greatest in the presence of sodium deoxycholate. 4. The Km for acceptor substrate was 1.6 x 10(-5)M in the reaction for dolichol phosphate mannose synthesis and 2.2 x 10(-5)M in the reaction for dolichol pyrophosphate N-acetylglucosamine formation. 5. The Km for GDP-mannose was 1.4 x 10(-5)M and for UDP-N-acetylglucosamine-6.2 x 10(-5)M. At saturating concentrations of donor substrates V values (pmol/min/mg) were 1330 and 150, respectively.     

Kinetic data for redox reactions of cytochrome c with Fe(CN)5X complexes and the question of association prior to electron transfer

Use of rigorous equilibration kinetics to evaluate rate constants for the Fe(CN)6 4- reduction of horse-heart cytochrome c in the oxidized form, cyt c (III), has shown that limiting kinetics do not apply with concentrations of Fe(CN)6 4- (the reactant in excess) in the range 2-10 x 10(-4) M, I = 0.10 M (NaCl). The reaction conforms to a first-order rate law in each reactant, and at 25 degrees C, pH 7.2 (Tris), it is concluded that K for association prior to electron transfer is less than 200 M-1. From previous studies at 25 degrees C, ph 7.0 (10(-1) M phosphate), I = 0.242 M (NaCl), a value K = 2.4 x 10(3) M-1 has been reported. Had such a value applied, some or all of the redox inactive complexes Mo(CN)8 4-, Co(CN)6 3-, Cr(CN)6 3-, Zr(C2O4)4 4- present in amounts 5-20 x 10(-4) M would have been expected to associate at the same site and partially block the redox process. No effect on rats was observed. With the reductants Fe(CN)5(4-NH2-py)3- and Fe(CN)5(imid)3-, reactions proceeded to greater than 90% completion and rate laws were again first order in each reactant. Rate constants (M-1 sec-1) at 25 degrees C, pH 7.2 (Tris), I = 0.10 M (NaCl), are Fe(CN)6 4- (3.5 x 10(4)), Fe(CN)5(4-NH2py)3- (6.7 x 10(5), and Fe(CN)5(imid)3- (4.2 x 10(5). Related reactions in which cyt c(II) is oxidized are also first order in each reactant, Fe(CN)6 3- (9.1 x 10(6)), Fe(CN)5(NCS)3- (1.3 x 10(6)), Fe(CN)5(4-NH2py)2- (3.8 x 10(6) at pH 9.4), and Fe(CN)5(NH3)2- (2.75 x 10(6) at ph 8). Redox inactive Co(CN)6 3- (1.0 x 10(-3) M) has no effect on the reaction of Fe(CN)6 3- which suggests that a recent interpretation for the Fe(CN)6 3- oxidation of cyt c(II), I = 0.07 M, may also require reappraisal.     

Size, Composition, and Structure of the Deoxyribonucleic Acid of Herpes Simplex Virus Subtypes 1 and 2

Studies of the size, composition, and structure of the deoxyribonucleic acid (DNA) of the F and G prototypes of herpes simplex virus (HSV) subtypes 1 and 2 (HSV-1 and HSV-2) showed the following. (i) As previously reported by Good-heart et al. HSV-1 and HSV-2 DNA have a buoyant density of 1.726 and 1.728 g/cm(3), corresponding to 67 and 69 guanine +/- cytosine moles per cent, respectively. The difference in guanine plus cytosine content of the DNA species was confirmed by the finding of a 1 C difference in T(m). (ii) The DNA from purified virus on cocentrifugation with T4 DNA in neutral sucrose density gradients sedimented at 55S, corresponding to 99 +/- 5 million daltons in molecular weight. HSV-1 and HSV-2 DNA could not be differentiated with respect to size. (iii) Cosedimentation of alkali-denatured DNA from purified virus with T4 DNA on alkaline sucrose density gradients consistently yielded several bands of single-stranded HSV DNA ranging from fragments 7 x 10(6) daltons to intact strands 48 x 10(6) daltons in molecular weight.     

Pathways in the binding and uptake of ferritin by hepatocytes

The binding and uptake of rat liver ferritin by primary cultures of rat liver hepatocytes was studied in order to assess the relative importance of saturable, high-affinity pathways and nonspecific processes in the incorporation of the protein by the cells. To minimize artifacts, ferritin not subjected to heat treatment and labeled in vivo with 59Fe was used. Binding to cell membranes was estimated from incubations performed at 4 degrees C. After 2 h, when a steady state in cell-associated ferritin had been achieved, approx. 4-10(4) binding sites per cell were observed, with an affinity constant for ferritin of 1 x 10(9) M-1. At 37 degrees C, the maximal uptake from these sites was 1.3 x 10(5) ferritin molecules/cell per h. For ferritin molecules bearing an average of 2400 iron atoms, this uptake amounts to 5 x 10(6) iron atoms/cell per min. Half-maximal uptake was achieved at a ferritin concentration, or KM1, of 3 x 10(-9) M. Although uptake rates at least a thousand times greater could be achieved by binding to the much larger number of low-affinity sites, the apparent KM2 for such 'nonspecific' uptake was 4 x 10(-7) M. At ferritin concentrations up to 2 nM, at least 90% of ferritin bound and taken up by hepatocytes involves saturable, high-affinity sites, presumably true ferritin receptors.     

Evaluation of equilibrium constants for the interaction of lactate dehydrogenase isoenzymes with reduced nicotinamide-adenine dinucleotide by affinity chromatography.

Rabbit muscle lactate dehydrogenase was subjected to frontal affinity chromatography on Sepharose-oxamate in the presence of various concentrations of NADH and sodium phosphate buffer (0.05 M, pH 6.8) containing 0.5 M-NaCl. Quantitative interpretation of the results yields an intrinsic association constant of 9.0 x 10 (4)M-1 for the interaction of enzyme with NADH at 5 degrees C, a value that is confirmed by equilibrium-binding measurements. In a second series of experiments, zonal affinity chromatography of a mouse tissue extract under the same conditions was used to evaluate assoication constants of the order 2 x 10(5)M-1, 3 x 10(5)M-1, 4 x 10(5)M-1, 7 x 10(5)M-1 and 2 x 10(6)M-1 for the interaction of NADH with the M4, M3H, M2H2, MH3 and H4 isoenzymes respectively of lactate dehydrogenase.     

The vasodilator effect of thiamylal in dog mesenteric artery: contribution of intracellular action.

The effects of thiamylal on contractions induced by various mechanisms were investigated in mesenteric arteries isolated from dogs. Thiamylal (10(-4) to 10(-3) M) significantly inhibited contractions induced by KCl (20 mM) in normal media, and those induced by norepinephrine (10(-5) M) in normal and Ca(2+)-free media. Caffeine-induced contraction was significantly inhibited by thiamylal in the concentrations greater than 3 x 10(-5) M in intact fibers and 10(-5) M in chemically skinned fibers. Chemically skinned fibers that were precontracted with Ca2+ were relaxed by thiamylal in concentrations lower than those required to relax intact fibers that were precontracted with KCl (20 mM); the ED50 was 1.52 x 10(-5) M in skinned fibers and 5.50 x 10(-4) M in intact fibers. These results suggest that intracellular mechanisms are involved in thiamylal-induced vasodilatation of dog mesenteric artery.     

Existence of two types of postjunctional alpha-adrenoceptors in the isolated canine internal carotid artery

The pharmacological characteristics of postjunctional alpha-adrenoceptors in isolated canine internal carotid arteries were investigated by the use of selective agonists and antagonists for alpha 1- and alpha 2-adrenoceptors. Norepinephrine, phenylephrine, and xylazine caused concentration-dependent contractions in the helical strips. The contraction induced by 10(-4)M xylazine was significantly smaller than that produced by 10(-4)M norepinephrine or 10(-4)M phenylephrine. The contraction induced by 10(-4)M phenylephrine was almost the same value as that induced by 10(-4)M norepinephrine. Phentolamine (10(-8) and 10(-7)M) caused a parallel shift to the right of the concentration-response curve to norepinephrine. The contractile responses to low concentrations of norepinephrine were significantly suppressed by pretreatment with an alpha 2-antagonist such as yohimbine (10(-9) and 10(-8)M) or DG5128 (10(-7) and 10(-6)M). On the other hand, the responses to higher concentrations of norepinephrine were mainly reduced by low concentrations of an alpha 1-antagonist, prazosin (3 x 10(-10) and 3 x 10(-9)M). These results suggest that both alpha 1- and alpha 2-adrenoceptors are located on the plasma membrane of smooth muscle cells in canine internal carotid arteries and that the norepinephrine-induced contractions at low and high concentrations are mainly mediated by activation of alpha 2- and alpha 1-adrenoceptors, respectively.     

Effect of procaine hydrochloride on the aggregation behavior and suspension viscoelasticity of human red blood cells

The present study examined the effects of procaine hydrochloride (PRHCL), a cationic local anesthetic, on the aggregation behavior of human red blood cells (RBC); the effects of PRHCL on RBC suspension viscoelasticity, cell shape, volume and density were also investigated. Four indices of RBC aggregation, induced by autologous plasma or 3 g% dextran T70, were evaluated by a computerized light transmission method, and the viscous and elastic components of the complex viscosity were determined by oscillatory viscometry. Low concentrations of PRHCL (8 x 10(-5) to 8 x 10(-4) M) significantly (p less than 0.05 or better) reduced the extent of aggregation (maximal decrease of 22% at 8 x 10(-4) M), but did not alter the viscoelastic components, cell shape, volume or density. The anti-aggregating effect of PRHCL (8 x 10(-4) M) in plasma significantly (p less than 0.005) decreased with time; this temporal effect was abolished by addition of eserine (1 x 10(-4) M). High concentrations of PRHCL (8 x 10(-2) M) caused: 1) increased extent of aggregation and decreased strength of the aggregates (p less than 0.01 or better); 2) elevation of both viscoelastic components for cells in plasma or buffer; 3) a discocyte-stomatocyte shape change; 4) decreased cell density (p less than 0.001) without alteration of cell volume. Our results at low concentrations of PRHCL suggest a mechanism based on an increase of RBC negative surface potential; at the highest concentration, the effects are most likely due to altered cell shape and deformability, and to decreased RBC negative surface potential.