Chenopod and amaranth pollen: electron-microscopic identification.

Electron-microscopic criteria for distinguishing chenopod and amaranth pollen have been found in the number of minute holes and spinules, and especially in the ratio between them, which is 1.5 in amaranths and 1.6 in chenopods. Sixty-eight percent of total fossil pollen from a Classic Maya level (carbon-14 age 1380 +/- 120 B.P.) in a lake-sediment core from El Salvador belongs to wild amaranths, which presumably invaded corn fields. Fossil chenopodiaceous pollen from a depth of 3.85 meters (about 4000 years old) at Tinte, the Netherlands, is mostly Atriplex littoralis, which was evidently very common on coastal marshes in the middle sub-Boreal period.

Successful therapeutic use of a single-dose of rituximab on relapse in adults with minimal change nephrotic syndrome.

Minimal change nephrotic syndrome (MCNS) usually is considered to have a good renal prognosis, but the frequency of relapses is a therapeutic challenge to physicians. The treatment of patients with multiple relapses remains a matter of controversy, because few controlled studies are available. We report the case of a 25-year-old man who experienced relapses of MCNS. Single-dose rituximab therapy (total dose 500 mg) was given during the fourth relapse. Complete remission occurred 10 days later, when no CD19/20-positive B cells were detected in the blood. This the first report of efficacy of single-dose rituximab therapy to treat multi-relapsing MCNS in an adult patient.

The relation between spike-timing dependent plasticity and Ca2+ dynamics in the hippocampal CA1 network.

In our previous study, spike timing dependent synaptic plasticity (STDP) was investigated in the CA1 area of rat hippocampal slices using optical imaging. It was revealed that the profiles of STDP could be classified into two types depending upon layer specific location along the dendrite. The first was characterized by a symmetric time window observed in the proximal region of the stratum radiatum (SR), and the second by an asymmetric time window in the distal region of the SR. Our methods involved the bath-application of bicuculline (GABA(A) receptor antagonist) to hippocampal slices, which revealed that GABAergic interneuron projections were responsible for the symmetry of a time window. In this study, the intracellular Ca2+ increase of hippocampal CA1 neurons, induced by the protocol of timing between pre- and post-synaptic excitation (i.e. STDP protocol), was measured spatially by using optical imaging to investigate how the triggering of STDP is dependent on intracellular calcium concentration. We found that the magnitude of STDP was closely related to the rate of Ca2+ increase ("velocity") of calcium transient during application of induction stimuli. Location dependency was also analyzed in terms of Ca2+ influx. Furthermore, it was shown that decay time constant of Ca2+ dynamics during the application of STDP-inducing stimuli was also significantly correlated with STDP.

Structural and functional analysis of a novel coiled-coil protein involved in Ypt6 GTPase-regulated protein transport in yeast.

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations. SYS3 encodes a 105-kDa protein with a predicted high alpha-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum-derived vesicles to the cis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in alpha-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.

Phase shift of subthreshold theta oscillation in hippocampal CA1 pyramidal cell membrane by excitatory synaptic inputs.

Hippocampal CA1 neurons receive multiple rhythmical inputs with relatively independent phases during theta activity. It, however, remains to be determined how these multiple rhythmical inputs affect oscillation properties in membrane potential of the CA1 pyramidal cell. In order to investigate oscillation properties in the subthreshold membrane potential, we generated oscillations in the membrane potential of the CA1 pyramidal cells in rat hippocampal slices in vitro with a sinusoidal current injection into the pyramidal soma at theta band frequencies (4-7 Hz), and analyzed effect of rhythmically excitatory synaptic inputs. The Schaffer collaterals were stimulated with a cyclic Gaussian stimulation method, whose pulse intervals were distributed at 10 pulses/cycle (5 cycles/s). We found that the cyclic Gaussian stimulations induced membrane potential oscillations and their phase delays from the mean of the pulse distribution were dependent on membrane potential oscillation amplitude. We applied four pairs of cyclic Gaussian stimulations and somatic sinusoidal current stimulations at the same frequency (5 Hz) with varying phase differences (-pi/2, 0, pi/2, pi rad). The paired stimulations induced phase distributions of the oscillation in the membrane potential, which showed a dependency on an increasing membrane potential oscillation amplitude response to cyclic Gaussian stimulation. This membrane potential dynamic was exhibited by the mixture of the membrane potential oscillation-amplitude-dependent phase delay and the linear summation of the two sinusoidal waves. These suggest that phases of the membrane potential oscillation are modulated by excitatory synaptic inputs. This phase-modulation by excitatory synaptic inputs may play a crucial role for memory operation in the hippocampus.

Heterogeneity of macrophages evidenced by variability in their glycoconjugates.

Staining rat tissues with a battery of 15 lectin-horseradish peroxidase conjugates showed that macrophages contain glycoconjugates possessing terminal alpha, beta-galactose, N-acetylgalactosamine, fucose, and N-acetylneuraminic acid, plus two terminal disaccharides. Dissimilar binding of lectins by different phagocyte populations in the same or different organs evidenced variability in glycoconjugates according to the location of the macrophages. With a group of four lectins, macrophages stained most intensely in lung, next strongest in splenic red pulp and lymph node sinuses, and weakest in skin and liver. Two populations of macrophages were newly recognized in spleen on the basis of content of fucose-rich glycoconjugate. These included a necklace-like band of macrophages at the border between marginal zone and germinal center and distinctive macrophages dispersed throughout the marginal zone. In lymph nodes, phagocytes stained strongly in the germinal centers and weakly in sinuses for glycoconjugate with N-linked oligosaccharides and conversely for glycoconjugate with terminal beta-galactose. Variable lectin binding indicated heterogeneity of thymic macrophages. Lectin cytochemistry offers increased sensitivity for detecting macrophages in tissue sections, provides selective staining that shows the prevalence and distribution of the phagocytes and differentiates macrophages into separate subtypes.

13-cis retinoic acid exerts its specific activity on human sebocytes through selective intracellular isomerization to all-trans retinoic acid and binding to retinoid acid receptors.

Despite its potent biologic effect on human sebocytes, 13-cis retinoic acid exhibits low binding affinity for cellular retinoic acid binding proteins and nuclear retinoid receptors. Hence, 13-cis retinoic acid may represent a pro-drug possibly acting through all-trans isomerization. In this study, marked isomerization of 13-cis retinoic acid has been confirmed in cultured SZ95 sebocytes showing 2- to 15-fold higher levels of all-trans retinoic acid at 12-72 h, as measured by high performance liquid chromatography. In contrast, only low amounts of all-trans retinoic acid were converted intracellularly to its 13-cis isoform. 9-cis retinoic acid was not detected after either 13-cis retinoic acid or all-trans retinoic acid treatment. The rapid isomerization of 13-cis retinoic acid to high levels of all-trans retinoic acid was a sebocyte-specific event, as no significant isomerization of 13-cis retinoic acid to all-trans retinoic acid occurred in HaCaT keratinocytes. De novo mRNA expression of cytochrome P450 1A1, a major xenobiotic metabolizing enzyme, in SZ95 sebocytes was induced by all-trans retinoic acid, but not by 13-cis retinoic acid. In addition, mRNA levels of cellular retinoic acid-binding protein II, which is supposed to regulate the concentration of intracellular all-trans retinoic acid, rapidly increased under all-trans retinoic acid treatment (30 min-6 h), whereas the 13-cis retinoic acid effect was markedly weaker and delayed. Both 13-cis retinoic acid and all-trans retinoic acid suppressed mRNA expression of cytochrome P450 1A2. In parallel experiments, 13-cis retinoic acid significantly reduced SZ95 sebocyte proliferation at 10-7 M, show- ing 30-40% inhibition after 9 d. All-trans retinoic acid and 9-cis retinoic acid exhibited similar anti-proliferative effects. AGN 193109, a pan-antagonist of the retinoic acid receptors, antagonized the anti-proliferative activity of all retinoic acid isomers tested in a concentration-dependent manner with complete abolishment at ratios of 1:10 13-cis retinoic acid and 1:1 all-trans retinoic acid. Coincubation of SZ95 sebocytes with 13-cis retinoic acid and AGN 193109 did not alter the intracellular concentration of 13-cis retinoic acid and its isomerization profile. In contrast, the retinoid X receptor antagonist CD 3507 did not affect the inhibition of SZ95 sebocyte proliferation induced by retinoic acids. Our findings indicate: (i) a selective 13-cis retinoic acid isomerization to all-trans retinoic acid in the intracellular compartment of SZ95 sebocytes; (ii) a reduced all-trans retinoic acid inactivation process after 13-cis retinoic acid treatment as compared with treatment with all-trans retinoic acid; and (iii) a retinoic acid receptor-mediated inhibition of SZ95 sebocyte proliferation. These data explain the sebocyte-specific activity of 13-cis retinoic acid and support a pro-drug/drug relation between 13-cis retinoic acid and all-trans retinoic acid.

Apg1p, a novel protein kinase required for the autophagic process in Saccharomyces cerevisiae.

Autophagic protein degradation includes bulk protein turnover with dynamic membrane reorganization, in which formation of novel organelles autophagosomes play key roles. We have shown that Saccharomyces cerevisiae performs the autophagy in the vacuole, a lytic compartment of yeast, in response to various kinds of nutrient starvation. Here we show that the APG1 gene, involved in the autophagic process in yeast, encodes a novel type of Ser/Thr protein kinase. Our results provide direct evidence for involvement of protein phosphorylation in regulation of the autophagic process. We found overall homology of Apglp with C. elegans Unc-51 protein, suggesting that homologous molecular mechanisms, conserved from unicellular to multicellular organisms, are involved in dynamic membrane flow.

Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae.

Autophagy in the yeast is similar to that in mammalian cells. A mutant designated as apg1 (autophagy) defective in accumulation of autophagic bodies in the vacuoles was isolated by selection using a light microscope from a mutagenized proteinase-deficient strain. In the apg1 strain, which has normal vacuolar proteinases, nitrogen starvation did not induce protein degradation. The apg1 mutant lost its viability faster than wild-type cells during nitrogen starvation. By using the loss of viability as a first screening test, 75 other apg mutants were selected. These apg mutants including apg1 fell into 15 complementation groups. Genetic analyses of representative apg mutants revealed that they all had single recessive chromosomal mutations. Strains with each apg mutation were defective in protein degradation in the vacuoles induced by nitrogen starvation and homozygous diploids for each apg mutation did not sporulate. These results on the apg mutants suggest that autophagy via autophagic bodies is indispensable for protein degradation in the vacuoles under starvation conditions, and that at least 15 APG genes are involved in autophagy in yeast.

Specific inhibition of pituitary prolactin production by energy restriction in C3H/SHN female mice.

Pituitaries were excised from control (C; 95 kcal/week) or energy restricted (ER; 48 kcal/week) female mice of 2, 3, 7, and 18 months of age. The total RNA and relative actin mRNA amounts in the pituitary were significantly greater in C than in ER mice both at 7 and 18 months. Prolactin (PRL) mRNA, standardized with actin mRNA, was significantly less in ER mice of 7 (50%) and 18 (51%) months of age than in age-matched controls, suggestive of specific inhibition of PRL mRNA transcription. Pituitary RNA and actin mRNA increased from 7 to 18 months in C mice but not in ER mice. Similarly, mean pituitary volumes increased between 2 and 18 months in C mice but not in ER mice. PRL mRNA, standardized with actin mRNA, did not change in either C or ER mice 7-18 months of age. All examined C mice of 3, 7, and 18 months of age had estrous cycles but none of the ER mice of the same ages. After 1 month of ER, the pituitary volumes and serum insulin concentrations in 2-month-old female mice were reduced. Thus net reduction of PRL mRNA per pituitary by ER is attributable to decreases in pituitary size and specific inhibition of PRL production, both of which may be due to low estrogen and insulin levels.

Electrophysiological evidence of an increase in cold tolerance of cardiac muscles in mice after energy restriction.

Life-prolonging energy restriction (ER) has been known to extend longevity. The heart was selected as the target organ of ER and the electrophysiological properties of ER on the heart were investigated. Action potential parameters were measured on ventricular papillary muscles of C57BL/6 mice (2-6 months of age). Resting membrane potential (Rm) did not change even when the temperature was lowered to 20 degrees C in ER mice (-67.5 +/- 0.8 mV), however, the membrane was depolarized in the control (-61.1 +/- 1.1 mV). Action potential duration measured at 30 and 50% repolarization was significantly prolonged in ER mice at 20-35 degrees C. Ouabain (10 microM) decreased Rm in ER mice at 20 degrees C (-68.6 +/- 1.0 to -63.6 +/- 0.8 mV), but failed to decrease Rm in the control (-60.6 +/- 1.8 to -62.1 +/- 1.2 mV). There were no significant differences in extracted Na, K-ATPase activity or affinity and binding capacity of ouabain between ER and control hearts. These results indicate that in ER mice the lack of effect of temperature on Rm was not due to a change in the physicochemical properties of Na, K-ATPase. The present study collectively suggests that ER increases cold tolerance in the heart of mice.

Increase in housing temperature can alleviate decrease in white blood cell counts after energy restriction in C57BL/6 female mice.

White blood cell (WBC) counts decrease after energy restriction (ER) in both rodents and humans. ER reduces cellular proliferation rates in various organs of rodents. Increases in housing temperatures can cancel this inhibitory effect of ER. We investigated whether a high housing temperature can alleviate decreases in WBC counts after ER, since peripheral WBC counts at basal levels are considered to represent cellular proliferation rates in the bone marrow. C57BL/6 female mice were allocated to three groups: the control (397 kJ/week), the ER (176 kJ/week) and the ER housed-in-incubator group (ERI: 134 kJ/week). The former two groups were housed at 21-23 degrees C and the third group was housed at 30 degrees C. At 4 and 13 months of age, WBC counts were significantly lower in ER or ERI mice than in control mice. WBC counts, however, were significantly higher in ERI mice (194%) than in ER mice at 13 months of age. A high housing temperature was proved to antagonize the effect of ER on WBC counts, as it antagonizes suppressive effects of ER on cellular proliferation rates in various organs.

Energy restriction suppresses microsomal Ca(2+)-ATPase activities in various organs in C57BL/6 female mice in both euthermic and torpor states.

C57BL/6 female mice were fed a daily control diet (n = 5, 3.9 g/day, 95 kcal/week) or ER diet (n = 5, 2.3 g/day, 48 kcal/week) at 1800 h from 6 weeks of age. Telemetry, conducted at 6 months of age, confirmed that all ER mice entered daily torpor (core body temperature less than 31 degrees C) for 6.63 +/- 2.34 h/day while control mice were euthermic (> 35 degrees C). In vitro activities of microsomal Ca(2+)-ATPase were determined in the brain, liver, salivary gland and kidney from these mice at 6 months of age. Assays were performed at three incubation temperatures of 37 degrees C, 31 degrees C and 25 degrees C. In assays at 37 degrees C, the activities of Ca(2+)-ATPase in the brain and salivary gland from ER mice were lower than those in corresponding organs from control mice. The suppression became profound as the incubation temperature decreased. On the other hand, at 37 degrees C Ca(2+)-ATPase activities in the liver and kidney from ER mice were not lower than those in corresponding organs from control mice, but decreased significantly at low temperatures. Microsomal Ca(2+)-ATPase activities thus appeared to be reduced in ER mice, although it remains unknown whether the present results represent reduced in vivo capacities to regulate cytosolic Ca2+ concentrations.

Caloric restriction perturbs the pituitary-ovarian axis and inhibits mouse mammary tumor virus production in a high-spontaneous-mammary-tumor-incidence mouse strain (C3H/SHN).

Dietary restriction (DR) retards aging and extends maximum life span. It is also known to decrease the incidence of hormone-dependent tumors. In the present investigation, we focussed primarily on the influence of DR on the pituitary-ovarian axis, and subsequently on gene expression of the mouse mammary tumor virus. F1 females from the mating of SHN female and C3H male mice were used in this study, since these hybrids display a very high incidence of mouse mammary tumors. The mice weaned at 3 weeks were raised on either a calorically-restricted diet (DR: 50 kcal/week; N = 5) or on a control diet (C: 95 kcal/week N = 5) for 5 weeks. Three C57BL/6J Jcl ad libitum-fed female mice, 8 weeks of age, were used as reference animals since this strain has a very low incidence of mammary tumors. The mean cellular contents of prolactin (PRL) and growth hormone (GH) in the pituitary, as determined by immunohistochemistry, were found to be reduced in mice raised on the DR diet. The decrease in the mean cellular content of PRL (50% of the mean control value) was accompanied by a decrease in the number of lactotrophs (17% of the mean value of control diet mice). However, the decrease in cellular content of GH (53% of the mean control value) was not accompanied by a decrease in the number of somatotrophs (no. of somatotrophs in DR = 103% of mean control value). Histologically, ovaries from DR mice showed many growing and atretic follicles, with few corpora lutea. In contrast, both control-diet and reference (C57BL/6J Jcl) mice showed two or three corpora lutea per ovarian section. In accordance with this finding, DR mice had not established stable estrus cycles by 8 weeks of age, in contrast to both control-diet and reference mice. Since caloric restriction has been shown to decrease mammary tumor virus (MMTV) gene expression, MMTV production was investigated by electron microscopy to confirm the validity of our experimental conditions. In DR or reference C57BL/6J Jcl mice, MMTV particles were rarely found in the mammary gland samples, but were always found in samples from control mice. The development of mammary glands, as indicated by the number of villi or the development of the rough endoplasmic reticulum, was delayed in DR mice. Thus, it was concluded that caloric restriction decreases the number of lactrotrophs, inhibits ovulation and delays mammary gland development. This immature status is considered to be due to perturbations in the pituitary-ovarian axis by caloric restriction.

Possible mechanism of acetaldehyde-induced noradrenaline release from sympathetic nerve terminals in isolated blood vessels.

1. Vasoconstrictor responses to acetaldehyde were investigated in isolated and perfused canine intermediate auricular (ear) arteries. 2. Single injections of small doses of acetaldehyde (1-3 mumol) induced vasoconstriction in a dose-related manner and showed no tachyphylaxis. On the other hand, large doses of acetaldehyde (10-30 mumol) frequently caused tachyphylaxis when injected at 10 min intervals. 3. After tyramine treatment, constrictions induced by a large dose of acetaldehyde were consistently restored temporarily. 4. The acetaldehyde-induced vasoconstriction was inhibited by bunazosin, a potent alpha 1-adrenoceptor antagonist. 5. A small dose of imipramine blocked tyramine-induced vasoconstriction, but had no significant influence on noradrenaline (NA)-induced constrictions, and caused slight potentiation of acetaldehyde-induced constrictions. 6. Hydrocortisone treatment did not modify tyramine-induced vasoconstrictions and slightly suppressed NA- and acetaldehyde-induced constrictions but not significantly. 7. It is suggested that acetaldehyde causes a release of NA from a NA store of the sympathetic nerve terminals which is different from the tyramine-sensitive NA store, and that the acetaldehyde-sensitive NA store may be readily filled up with NA from the tyramine-sensitive store.

Physico-chemical properties of silk fibroin membrane as a biomaterial.

A water-insoluble silk fibroin membrane was prepared by immersing a silk fibroin membrane as cast in 50 vol% aqueous methanol solution for different periods of time at 25 degrees C. To use the membrane as a biomaterial, oxygen and water vapour permeability, transparency, mechanical property and enzymatic degradation behaviour in vitro of the membrane in the wet state were investigated. These physico-chemical properties changed according to the condition of the methanol treatment. The membrane had oxygen permeability, water vapour permeability, transparency and biodegradability.

Synthesis of poly(ethylene glycol)-silk fibroin conjugates and surface interaction between L-929 cells and the conjugates.

Poly(ethylene glycol) (PEG)-silk fibroin (SF) conjugates (PEG2-SF) were prepared by the chemical modification of solubilized SF with 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine (actPEG2) in borate buffer at 37 degrees C. The IR spectra and DSC curves of PEG2-SF and SF suggested the introduction of PEG into SF by the modification and the beta-sheet structure of both SF and PEG2-SF induced by the treatment with methanol aqueous solutions. The content of the PEG component in PEG2-SF was evaluated to be 67% by weight from the melting enthalpy change of PEG observed on the DSC thermogram of PEG2-SF. Water content and contact angle measurements of SF before and after the modification indicated that the hydrophilicity of the PEG2-SF surface increased compared with that of SF. The attachment and growth of fibroblast cells (L-929) on the matrix of PEG2-SF were studied by a cell culture method. PEG2-SF exhibited very low cell attachment and growth, though SF exhibited high cell attachment and growth. The filopodium of the cells attached to PEG2-SF could not be found, and the cells aggregated to form masses in scanning electron microscopy images. These results could be explained in terms of the increased hydrophilicity of the PEG2-SF surface.

Treatment of standard risk acute lymphoblastic leukemia in children with the Tokyo Children Cancer Study Group (TCCSG) L84-11 protocol in Japan.

During the period June 1984 through February 1989, 207 children, newly diagnosed with standard-risk acute lymphoblastic leukemia (ALL), were treated with the Tokyo Children Cancer Study Group L84-11 protocol. The patients were randomized into two regimen groups (S1 and S2). All patients received identical induction therapy with vincristine, prednisolone, and L-asparaginase. For central nervous system (CNS) prophylaxis, the patients in regimen S1 received cranial irradiation (18 Gy) and intrathecal chemotherapy starting at 5 weeks, while those in regimen S2 received this therapy starting at 24 weeks. Consolidation consisted of cyclic therapy with vincristine and dexamethasone, and then, after 128 weeks, with medium-dose methotrexate, CNS chemoprophylaxis, and dexamethasone. Regimen S2 employed early consolidation with three doses of medium-dose methotrexate and CNS chemoprophylaxis. The treatment duration was 3.5 years. Complete remission was achieved in 96.0% and 99.0% of the patients in regimens S1 and S2, respectively. Event-free survival (EFS) rates were 80.0 +/- 3.5% in total, 74.5 +/- 5.7% in regimen S1, and 85.1 +/- 5.7% in regimen S2 at a median follow-up of 42 months (< 85 months). There was no significant difference in the EFS between the two regimens. As compared with recently reported results of treatment for this disease, our protocol can yield similar EFS rates with mild toxicity.

Spike trains in rat periaqueductal gray depend on the stochastic properties of interacting electrical stimulation trains.

Spike trains flowing into the periaqueductal gray (PAG) might be discriminated from one another by PAG neurons on the basis of the distribution or sequence of their respective interspike intervals. The various sequences of interspike intervals characteristic of spontaneous PAG unit activities were assessed in a preliminary experiment. These sequences were then simulated by means of appropriate mathematical functions. These functions allowed the production of stimulation trains that were applied to two PAG sites to induce spike trains with similar sequences in order to reveal the sensitivity of PAG neurons to the stochastic structure of afferent spike trains. We placed emphasis on parameters of the spike train that proved to be altered independently of any alteration of the corresponding parameters in the stimulation train. The mean pulse rate is the simplest example of such a parameter as it was never altered in the stimulation train. Alterations of either the distribution or sequence of pulses in the stimulation train were found to affect the mean discharge rate in a number of cases (30-40% of the cases). Despite their moderate degree (20-30% mean rate alteration) such differential effects could correspond to stimulation-induced differential behavioral effects as was shown in a previous study. Furthermore, a specific dependence of the generated spike trains on the sequential structure of the stimulation train was observed in some cases when appropriate stimulation trains were simultaneously applied to another PAG stimulation site. This fact is worth considering in relation to the integrative function of the PAG neuronal network.

Predominant acetylcholine-induced vasoconstriction in isolated, perfused simian facial veins.

Using the cannula insertion method, we investigated the vascular response to acetylcholine (ACh) and other vasoactive substances. ACh consistently induced only vasoconstriction, whereas isoproterenol and norepinephrine usually induced dilatation. Vasoconstriction induced by phenylephrine was less potent than that induced by ACh. Clonidine and xylazine did not induce significant vascular responses. ACh-induced constrictions were readily inhibited by atropine and slightly potentiated by physostigmine. They were slightly but significantly inhibited by pirenzepine (a muscarinic M1-receptor antagonist), but not influenced by AF-DX 116 (a M2-receptor antagonist). 4-DAMP (4-diphenylacetoxy N-methylpiperidine; a M3-receptor antagonist), strongly inhibited the ACh-induced constrictions. They were not modified by bunazosin but slightly suppressed by diltiazem. Removal of the endothelium did not significantly modify the ACh-induced constrictions. From our results, we conclude that the simian facial vein has many constrictory muscarinic receptors, especially of the M3 subtype.