Biomedical subjects

E Hamel

Publications and source records attributed to E Hamel.

At least 163 records · Page 9Linked to original sources

PubMed1992

Natural products which interact with tubulin in the vinca domain: maytansine, rhizoxin, phomopsin A, dolastatins 10 and 15 and halichondrin B.

This paper summarizes published data on the interactions of tubulin with antimitotic compounds that inhibit the binding of vinca alkaloids to the protein. These are all relatively complex natural products isolated from higher plants, fungi and marine invertebrate animals. These agents are maytansine, rhizoxin, phomopsin A, dolastatins 10 and 15 and halichondrin B and their congeners. Effects on tubulin polymerization, ligand binding interactions and structure-activity relationships are emphasized.

Animals↗

PubMed1992

Identification of multiple muscarinic binding site subtypes in cat and human cerebral vasculature.

The binding characteristics of the nonselective muscarinic antagonist [3H]N-methyl scopolamine ([3H]NMS) have been studied in membrane fractions of cat and human cerebral blood vessels. A computer-fitting method was used to analyze the data obtained from association/dissociation, saturation and competition experiments. Specific binding of [3H]NMS to membrane preparations from cat and human pia-arachnoid vessels was found to be saturable (respective Bmax values of 98 +/- 15 and 67 +/- 7 fmol/mg protein) and of high affinity (KD values of 165 +/- 28 and 125 +/- 12 pM, respectively). Competition studies, in the presence of various well-characterized M1, M2 or M3 putative muscarinic antagonists, performed against the binding of [3H]NMS, revealed the heterogeneity of muscarinic binding sites in these vascular tissues. A population of M1 sites was clearly identified in both human and cat pial vessel membranes and accounted for approximately 40 (human) and 20% (cat) of the total population of cerebrovascular muscarinic binding sites labeled with [3H]NMS. Such observation was further supported by saturation studies of [3H]NMS binding performed under M1 blocking conditions (75 nM pirenzepine). Competition and saturation (in the presence of M2 antagonists) studies suggested the presence of M2 sites (approximately 35% of total sites) in cat pial vessels. However, under the same conditions, no M2 binding sites could be detected in human cerebrovascular membranes. A small population of M3 sites (approx. 20%) was found in both human and cat cerebrovascular membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

PubMed1991

Halichondrin B and homohalichondrin B, marine natural products binding in the vinca domain of tubulin. Discovery of tubulin-based mechanism of action by analysis of differential cytotoxicity data.

Data generated in the new National Cancer Institute drug evaluation program, which is based on inhibition of cell growth in 60 human tumor cell lines, were used to compare new compounds with agents of known mechanism of action in terms of their differential cytotoxicity. Two marine natural products, halichondrin B and homohalichondrin B, appeared repeatedly when the data base was probed with known antimitotic agents. We confirmed that both compounds were highly cytotoxic (IC50 values for L1210 murine leukemia cells of 0.3 and 1 nM, respectively), with accumulation of cells arrested in mitosis at toxic concentrations, that both inhibited the polymerization of purified tubulin, and that both inhibited microtubule assembly dependent on microtubule-associated proteins. Limited amounts of homohalichondrin B, the less active agent, were available, so only halichondrin B was studied in detail. Halichondrin B did not interfere with colchicine binding to tubulin, but it was a noncompetitive inhibitor of the binding of vinblastine to tubulin (apparent Ki, 5.0 microM). Halichondrin B was therefore compared with other agents which interfere with the binding of vinca alkaloids to tubulin (vinblastine, maytansine, dolastatin 10, phomopsin A, rhizoxin) in terms of its effects on tubulin polymerization, inhibition of GTP hydrolysis, inhibition of nucleotide exchange, and stabilization of tubulin, as well as the quantitative assessment of its effects on vinca alkaloid binding and inhibition of cell growth. Since halichondrin B was originally isolated from the same organism as the phosphatase inhibitor okadaic acid, and since it is about 50-fold more effective than okadaic acid as an inhibitor of L1210 cell growth, perturbations of cellular microtubules observed following treatment with okadaic acid should be interpreted cautiously.

Animals↗

PubMed1991

Vasocontractile muscarinic M1 receptors in cat cerebral arteries: pharmacological identification and detection of mRNA.

The nature of the muscarinic receptor subtype mediating the acetylcholine (ACh)-induced constriction of the cat middle cerebral artery was investigated in vitro by recording the smooth muscle isometric tension of precontracted endothelium-denuded arterial segments. The ability of selective (pirenzepine, UH-AH 371, AF-DX 116, methoctramine, AQ-RA 741, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and hexahydro-sila-difenidol (HHSiD)) and non-selective (atropine) antagonists to inhibit the constriction elicited by ACh was estimated. In addition, using a subtype-specific ribonucleotide probe directed against mRNA encoding the human m1 (Hm1) muscarinic receptor, identification of the corresponding vascular receptor was undertaken in total RNA extracts from cat cerebral blood vessels. The potent inhibition of the ACh-induced constriction by M1 antagonists (pirenzepine and UH-AH 371; pA2 values respectively of 8.08 and 8.64), together with lower affinities of M2 (AF-DX 116; pA2 = 6.50, methoctramine; pA2 = 6.27 and AQ-RA 741; pA2 = 7.60) and M3 compounds (4-DAMP and HHSiD; with pA2 values of 8.85 and 7.76, respectively) strongly suggested the involvement of a pharmacological M1 receptor in this vasomotor response. Furthermore, Northern blot hybridization with the selective Hm1 ribonucleotide probe showed the presence of mRNA transcripts for this muscarinic receptor subtype in the cat cerebrovascular bed. The results indicate that muscarinic constriction in the feline cerebrovascular bed is mediated by a pharmacological M1 receptor subtype and that the corresponding m1 receptor mRNA is present in cat cerebral blood vessels. These findings clearly point to a role of M1 muscarinic receptors in cerebrovascular function.

Acetylcholine↗

PubMed1991

Hypercapnia and stimulation of the substantia innominata increase rat frontal cortical blood flow by different cholinergic mechanisms.

This study was designed to further investigate in the rat how the cerebrovascular response to excitation of the projections from the substantia innominata (SI) to the frontal cerebral cortex was mediated. Local cortical blood flow (CoBF) (by helium clearance) and tissue gas partial pressures (pO2, pCO2) (indices of energy metabolic activity) were measured in the frontal cortex in unanesthetized adult Fischer rats in response to electrical stimulation of the SI and, for comparison, in hypercapnic conditions. SI stimulation and hypercapnia increased CoBF to a similar extent (+92% and +106%, respectively). Differences between the changes in tissue gas partial pressures under hypercapnia and SI stimulation suggest that different patterns of flow-metabolism coupling prevail in the mechanisms underlying the two cerebrovascular responses. Cortical pCO2 increased under hypercapnia, but decreased during SI stimulation, indicating that a 'vascular' mechanism (i.e. independent of energy metabolism activation) is at least partly responsible for the flow increase in the latter condition. However, cortical pO2 rose more under hypercapnia than during SI stimulation, suggesting that oxygen consumption, and hence energy metabolism, was increased in the latter case. The ability of the acetylcholine esterase inhibitor physostigmine and the muscarinic receptor antagonist scopolamine to modulate the responses was quantified. In both experimental conditions, CoBF changes were potentiated by 0.15 mg/kg/h physostigmine (by factors of about 2). In contrast, 1 mg/kg scopolamine reduced by 65% the frontal CoBF response elicited by SI stimulation but was without effect on the response to hypercapnia. Thus, although a cholinergic mechanism may be implicated in both responses, activation of muscarinic receptors appears to occur when the stimulation originates from the SI but not from the hypercapnia.

Animals↗

PubMed1991

Growth suppression of transformed cells by a human placental extract not related to transforming growth factor beta.

We have examined whether human placental extracts contain tumour-growth-inhibitory factors. One fraction (EAP) from such extracts inhibited growth, in soft agar, of Ha-ras-transformed BALB/c 3T3 cells and human squamous lung carcinoma A-2182 cells. However, this fraction had no effect on the anchorage-dependent growth of these cells, although there was a slight mitogenic activity on nontransformed cells. These data together with those on plating efficiency indicated no significant cytotoxicity of EAP on transformed cell lines. Although this fraction contained transforming growth factor beta (TGF beta), this cannot account for its inhibitory activity, since (a) pure TGF beta does not inhibit anchorage-dependent growth of Ha-ras-transformed BALB/c 3T3 cells, (b) EAP retains its inhibitory activity in the presence of antibodies against TGF beta and (c) the inhibitory activity did not copurify with TGF beta. Partial characterization of our inhibitory factor suggests that the inhibitory factor is a new tumour-growth-inhibitory factor.

Animals↗

PubMed1991

Highly variable effects of beryllium and beryllium fluoride on tubulin polymerization under different reaction conditions: comparison of assembly reactions dependent on microtubule-associated proteins, glycerol, dimethyl sulfoxide, and glutamate.

Carlier et al. (1988, Biochemistry 27, 3555-3559; 1989, Biochemistry 28, 1783-1791) described enhancement of tubulin polymerization and stabilization of glycerol-induced microtubules by BeF3- (by addition of both BeSO4 and NaF to reaction mixtures). We were able to confirm the stabilization of glycerol-induced polymer reported by these workers, provided Mg2+ was also present in the reaction. When we examined polymerization dependent on microtubule-associated proteins (MAPs), however, we obtained very different results. BeF3- had no significant effect on this reaction, or the polymer formed, under any condition examined. Lower concentrations of BeSO4 alone, in contrast to a negligible effect in glycerol, enhanced polymerization with MAPs provided the concentrations of both Mg2+ and GTP were low; and Be2+ stabilized the polymer, if the GTP concentration was low, at both low and high Mg2+ concentrations. Higher concentrations of BeSO4 precipitated tubulin, an effect which was not affected by Mg2+, partially prevented but not reversed by MAPs, and prevented or reversed by either NaF or nucleotides at adequate concentrations. These results suggest that Be2+ binds at site(s) distinct from Mg2+ site(s), and that partial occupancy of these site(s) at lower Be2+ concentrations enhances tubulin polymerization and polymer stability, while extensive occupancy at higher Be2+ concentrations results in tubulin precipitation. Effects of Be2+ and BeF3- on polymerization dependent on dimethyl sulfoxide or glutamate were also evaluated. The dimethyl sulfoxide system displayed properties similar to those of the glycerol system, while the glutamate system was similar to the MAPs system.

Animals↗

PubMed1991

Synthesis and evaluation of stilbene and dihydrostilbene derivatives as potential anticancer agents that inhibit tubulin polymerization.

An array of cis-, trans-, and dihydrostilbenes and some N-arylbenzylamines were synthesized and evaluated for their cytotoxicity in the five cancer cell cultures A-549 lung carcinoma, MCF-7 breast carcinoma, HT-29 colon adenocarcinoma, SKMEL-5 melanoma, and MLM melanoma. Several cis-stilbenes, structurally similar to combretastatins, were highly cytotoxic in all five cell lines and these were also found to be active as inhibitors of tubulin polymerization. The most active compounds also inhibited the binding of colchicine to tubulin. The most potent of the new compounds, both as a tubulin polymerization inhibitor and as a cytotoxic agent, was (Z)-1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethene (5a). This substance was almost as potent as combretastatin A-4 (1a), the most active of the combretastatins, as a tubulin polymerization inhibitor. Compound 5a was found to be approximately 140 times more cytotoxic against HT-29 colon adenocarcinoma cells and about 10 times more cytotoxic against MCF-7 breast carcinoma cells than combretastatin A-4. However, 5a was found to be about 20 times less cytotoxic against A-549 lung carcinoma cells, 30 times less cytotoxic against SKMEL-5 melanoma cells, and 7 times less cytotoxic against MLM melanoma cells than combretastatin A-4. The relative potencies 5a greater than 8a greater than 6a for the cis, dihydro, and trans compounds, respectively, as inhibitors of tubulin polymerization are in agreement with the relative potencies previously observed for combretastatin A-4 (1a), dihydrocombretastatin A-4 (1c), and trans-combretastatin A-4 (1b). The relative potencies 5a greater than 8a greater than 6a were also reflected in the results of the cytotoxicity assays. Structure-activity relationships of this group of compounds are also discussed.

Antineoplastic Agents↗

PubMed1991

Acetylcholine levels and choline acetyltransferase activity in rat cerebrovascular bed after uni- or bilateral sphenopalatine ganglionectomy.

Endogenous acetylcholine (ACh) levels and choline acetyltransferase (ChAT) activity were measured in several vascular segments (major cerebral arteries, cortical pial vessels, and peripheral arteries) and nervous tissues [including the sphenopalatine ganglion (SPG)] in the rat. The effects of uni- or bilateral surgical ablation of the SPG, a putative origin of the cholinergic cerebrovascular innervation, were investigated on these two specific cholinergic markers at various postoperative times. ChAT activity and ACh levels were enriched in the cerebral as compared to the peripheral arteries. Among the cerebrovascular tissues tested, ACh levels were particularly high in the circle of Willis and the vertebrobasilar segments and, to a lesser extent, in the middle cerebral artery. Lower levels were found in the small pial vessels and choroid plexus. Overall, ChAT activity measured in different arterial beds paralleled the distribution of ACh. Following uni- or bilateral removal of the SPG, slight reductions (18-36%, statistically not significant) were observed in ChAT activity in rostral cerebral arteries and pial vessels overlying the frontal cortex. Similarly, bilateral ganglionectomy resulted in minor decreases (11-22%, not significant) in the cerebrovascular contents of ACh in these same vascular segments. These results clearly show that the SPG does not or only partly contributes to the cholinergic fibers that supply the cerebrovascular bed.

Acetylcholine↗

PubMed1991

Contractile 5-HT1 receptors in human isolated pial arterioles: correlation with 5-HT1D binding sites.

1. The 5-hydroxytryptamine (5-HT) receptor responsible for inducing vasoconstriction in human isolated pial arterioles has been pharmacologically characterized. 2. Of several 5-HT agonists tested, 5-carboxamidotryptamine (5-CT) was the most potent and the rank order of agonist potency can be summarized as: 5-CT greater than 5-HT greater than RU 24969 = alpha-methyl-5-HT = methysergide much greater than MDL 72832 = 2-methyl-5-HT much greater than 2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydro-naphthalene (8-OH-DPAT). With few exceptions, the maximal contractile responses of these agonists were comparable to that induced by 5-HT. 3. A correlation analysis performed between the agonists vascular potency (pD2 values) and their affinities (pKD values) published at various subtypes of 5-HT binding sites showed a positive significant correlation with rat cortical 5-HT1B (r = 0.86; P less than 0.01) and human caudate 5-HT1D (r = 0.98; P less than 0.005) subtypes. 4. Selective antagonists at 5-HT2 (ketanserin, mianserin, MDL 11939) and 5-HT3 (MDL 72222) sites were totally devoid of inhibitory activity on the 5-HT-induced contraction, an observation which agreed with the agonist data and further excluded activation of these receptors. In contrast, the 5-HT1-like/5-HT2 antagonist methiothepin and the non-selective 5-HT1D compound metergoline inhibited with high affinity the contraction induced by 5-HT with respective pA2 values of 8.55 +/- 0.16 and 6.88 +/- 0.05. This contractile response was, however, insensitive to 5-HT1B (propranolol) and 5-HT1C (mesulergine, mianserin) antagonists. 5. It is concluded that a 5-HT1-like receptor, which shares strong similarities with the 5-HTID binding sites identified in human caudate membranes, is mediating the vasocontractile action of 5-HT in human pial arterioles.

Arterioles↗

PubMed1991

Investigation of the mechanism of the interaction of tubulin with derivatives of 2-styrylquinazolin-4(3H)-one.

A new class of antimitotic agents, derivatives of 2-styrylquinazolin-4(3H)-one (SQZ), was recently described [J. Med. Chem. 33:1721-1728 (1990)]. Because they appeared to interact at a new ligand binding site on tubulin, we attempted to determine their mechanism of action as inhibitors of tubulin polymerization. Although in initial studies inhibition of colchicine binding was negligible, substantial and competitive inhibition of this reaction could be demonstrated with very short incubation times (less than 5 min), provided that a relatively low colchicine to tubulin ratio was used. The initial apparent failure to inhibit colchicine binding resulted from extremely rapid binding to tubulin and dissociation from tubulin by the SQZ derivatives, in comparison with the slow, temperature-dependent, poorly reversible binding of colchicine. The most inhibitory of the SQZ derivatives in the colchicine binding assay was 6-methyl-2-styrylquinazolin-4(3H)-one (NSC 379310), and its interaction with tubulin, particularly as an inhibitor of colchicine binding, was compared with that of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone (MTPT), because the binding parameters of MTPT with tubulin have been well described. The data indicate that NSC 379310 binds to tubulin and dissociates from the protein about 3 times as rapidly as MTPT. The other SQZ derivatives with equal or greater potency as inhibitors of tubulin polymerization but apparently less potency as inhibitors of colchicine binding presumably bind to and/or dissociate from tubulin even more rapidly than does NSC 379310.

Antineoplastic Agents↗

PubMed1990

Structure-activity studies with chiral isomers and with segments of the antimitotic marine peptide dolastatin 10.

Eighteen configurational isomers of the antimitotic peptide dolastatin 10 (Bai et al., Biochem Pharmacol 39: 1941-1949, 1990) derived from Dolabella auricularia, together with segments obtained as precursors in its synthesis (Pettit et al., J Am Chem Soc 111: 5463-5465, 1989), were examined as inhibitors of tubulin polymerization and as inhibitors of growth of L1210 murine leukemia cells in culture. Dolastatin 10 consists of four amino acids (in order from the amino terminus: dolavaline, valine, dolaisoleucine, and dolaproine), three unique to D. auricularia, linked to an unusual primary amine (dolaphenine, probably derived from phenylalanine) at what would otherwise be its carboxyl terminus. Dolastatin 10 has nine asymmetric carbon atoms, and available isomers included alternate configurations at five positions (positions 9 and 10 in the dolaproine moiety and positions 18, 19 and 19a in the dolaisoleucine moiety). For tubulin polymerization, only alterations at positions 18 and 19 resulted in loss of inhibitory activity of the isomer. In addition, a tripeptide containing dolavaline, valine and dolaisoleucine with all asymmetric carbons identical configurationally to those in dolastatin 10 was found to be about 30% as effective as dolastatin 10 in inhibiting tubulin polymerization. Cytotoxic effects were much more sensitive to alterations in the dolastatin 10 structure. The only modification which did not lead to reduced cytotoxicity was reversal of configuration at position 19a in the dolaisoleucine moiety. Both this isomer and dolastatin 10 had IC50 values of less than 1 nM. Several other isomers had IC50 values with the L1210 cells in the range of 30-90 nM, but these did not correlate well with their inhibitory effects on tubulin polymerization. The tripeptide effective as an inhibitor of tubulin polymerization had no activity against the L1210 cells.

Animals↗

PubMed1990

Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites.

Dolastatin 10, a potent antimitotic peptide from a marine animal, strongly inhibits microtubule assembly, tubulin-dependent GTP hydrolysis, and the binding of vinca alkaloids to tubulin. In studies of the binding of [3H]vincristine to the protein, with vinblastine as a control for competitive inhibition (Ki, 6.6 microM), we found that the macrolide antimitotic agents maytansine and rhizoxin were also competitive inhibitors (Ki values, 3.1 and 12 microM). Dolastatin 10 and an unrelated peptide antimitotic, phomopsin A, were more potent but noncompetitive inhibitors (Ki values, 1.4 and 2.8 microM). Since maytansine and, to a much lesser extent, vinblastine interfere with nucleotide exchange on tubulin, all drugs were examined for effects on nucleotide interactions at the exchangeable GTP site. Rhizoxin had effects intermediate between those of vinblastine and maytansine. Both peptides inhibited binding of radiolabeled GTP to tubulin even more strongly than did maytansine, but no drug displaced nucleotide from tubulin. The drugs were evaluated for stabilizing effects on the colchicine binding activity of tubulin. The peptides prevented loss of this activity, and vinblastine provided partial protection, while rhizoxin and maytansine did not stabilize tubulin. A tripeptide segment of dolastatin 10 also effectively inhibits tubulin polymerization and GTP hydrolysis. The tripeptide did not significantly inhibit either vincristine binding or nucleotide exchange, nor did it stabilize colchicine binding. These findings are rationalized in terms of a model with two distinct drug binding sites in close physical proximity to each other and to the exchangeable GTP site on beta-tubulin.

Animals↗

PubMed1990

Serotonin-synthesizing nerve fibers in rat and cat cerebral arteries and arterioles: immunohistochemistry of tryptophan-5-hydroxylase.

Nerve fibers synthesizing 5-hydroxytryptamine (5-HT or serotonin) were demonstrated in rat and cat cerebral blood vessels by immunohistochemical localization of the rate-limiting enzyme catalyzing the biosynthesis of 5-HT, namely tryptophan-5-hydroxylase (TPOH). TPOH-immunoreactive fibers were present in all parts of the circle of Willis with a somewhat less intense distribution in the vertebro-basilar segment. Single, small nerve fibers intermingled around the vessel wall were found in both species but clear TPOH-immunoreactive varicosities were observed predominantly in the rat. The most striking observation was the dense network of TPOH-positive fibers innervating some but not all small pial arteries, and, to a lesser extent, pial arterioles. These results provide the evidence that, at least in these species, the cerebrovascular bed is innervated by authentic serotonergic fibers which can synthesize their own 5-HT.

Animals↗

PubMed1990

N-acetylcolchinol O-methyl ether and thiocolchicine, potent analogs of colchicine modified in the C ring. Evaluation of the mechanistic basis for their enhanced biological properties.

Two colchicine analogs with modifications only in the C ring are better inhibitors than colchicine of cell growth and tubulin polymerization. Radiolabeled thiocolchicine (with a thiomethyl instead of a methoxy group at position C-10) and N-acetylcolchinol O-methyl ether (NCME) (with a methoxy-substituted benzenoid instead of the methoxy-substituted tropone C ring) were prepared for comparison with colchicine. Scatchard analysis indicated a single binding site with KD values of 1.0-2.3 microM. Thiocolchicine was bound 2-4 times as rapidly as colchicine, but the activation energies of the reactions were nearly identical (18 kcal/mol for colchicine, 20 kcal/mol for thiocolchicine). NCME bound to tubulin in a biphasic reaction. The faster phase was 60 times as fast as colchicine binding at 37 degrees C, and a substantial reaction occurred at 0 degrees C. The rate of the faster phase of NCME binding changed relatively little as a function of temperature, so the activation energy was only 7.0 kcal/mol. Dissociation reactions were also evaluated, and at 37 degrees C the half-lives of the tubulin-drug complexes were 11 min for NCME, 24 h for thiocolchicine, and 27 h for colchicine. Relative dissociation rates as a function of temperature varied little among the drug complexes. Activation energies for the dissociation reactions were 30 kcal/mol for thiocolchicine, 27 kcal/mol for NCME, and 24 kcal/mol for colchicine. Comparison of the activation energies of association and dissociation yielded free energies for the binding reactions of -20 kcal/mol for NCME, -10 kcal/mol for thiocolchicine, and -6 kcal/mol for colchicine. The greater effectiveness of NCME and thiocolchicine as compared with colchicine in biological assays probably derives from their more rapid binding to tubulin and the lower free energies of their binding reactions.

Animals↗

PubMed1990

Dolastatin 10, a powerful cytostatic peptide derived from a marine animal. Inhibition of tubulin polymerization mediated through the vinca alkaloid binding domain.

Dolastatin 10, a cytostatic peptide containing several unique amino acid subunits, was isolated from the marine shell-less mollusk Dolabella auricularia (Pettit GR, Kamano Y, Herald CL, Tuinman AA, Boettner FE, Kizu H, Schmidt JM, Baczynskyj L, Tomer KB and Bontems RJ, J Am Chem Soc 109: 6883-6885, 1987). Since our preliminary studies demonstrated that dolastatin 10 inhibited tubulin polymerization and the binding of radiolabeled vinblastine to tubulin, an initial characterization of the properties of dolastatin 10 included a comparison to other antimitotic drugs interfering with vinca alkaloid binding to tubulin (vinblastine, maytansine, rhizoxin, and phomopsin A). Dolastatin 10 inhibited the growth of L1210 murine leukemia cells in culture, with a concordant rise in the mitotic index, and its IC50 value for cell growth was 0.5 nM. Comparable values for the other drugs were 0.5 nM for maytansine, 1 nM for rhizoxin, 20 nM for vinblastine, and 7 microM for phomopsin A. IC50 values were also obtained for the polymerization of purified tubulin in glutamate: 1.2 microM for dolastatin 10, 1.4 microM for phomopsin A, 1.5 microM for vinblastine, 3.5 microM for maytansine, and 6.8 microM for rhizoxin. Dolastatin 10 and vinblastine were comparable in their effects on microtubule assembly dependent on microtubule-associated proteins. Preliminary studies indicated that dolastatin 10, like vinblastine, causes formation of a cold-stable tubulin aggregate at higher drug concentrations. We confirmed that rhizoxin, phomopsin A, and maytansine also inhibit the binding of radiolabeled vinblastine and vincristine to tubulin. Dolastatin 10 and phomopsin A were the strongest inhibitors of these reactions, and rhizoxin the weakest. Dolastatin 10, phomopsin A, maytansine, vinblastine, and rhizoxin all inhibited tubulin-dependent GTP hydrolysis. The greatest inhibition of hydrolysis was observed with dolastatin 10 and phomopsin A, and the least inhibition with rhizoxin.

Amino Acid Sequence↗

PubMed1990

Small pial vessels, but not choroid plexus, exhibit specific biochemical correlates of functional cholinergic innervation.

In an attempt to provide the biochemical foundations for a putative cholinergic innervation of small pial vessels and choroid plexus, we have assessed their ability to specifically accumulate choline, synthesize and release acetylcholine (ACh) in response to depolarization. Our results show that both small pial vessels and choroid plexus avidly accumulate choline via a sodium-dependent mechanism which could be inhibited by hemicholinium-3 (IC50 in pial vessels = 47.8 microM). Light microscopic examination of radioautographs from vessels incubated with [3H]choline revealed two distinct sites of accumulation in the vessel wall. One site probably corresponded to nerve terminals and the other was closely associated with the endothelial cells. In small pial vessels, a major proportion (60%-70%) of the choline acetyltransferase (ChAT) activity could be inhibited by 4-naphthylvinylpyridine (4-NVP), a potent inhibitor of neuronal ChAT; and, following either K+ or veratridine depolarization, a Ca2(+)-dependent release of authentic [3H]ACh could be measured. In contrast, the choroid plexus exhibited a rather low ChAT activity which was not inhibited by 4-NVP and no release of ACh could be detected in this tissue following depolarization. Altogether, the results of the present study show that (1) small pial vessels exhibit all the most selective biochemical markers that are characteristic of cholinergic nerves; (2) [3H]choline in pial vessels can be accumulated in non-neuronal elements which probably correspond to the endothelial cells; and (3) the choroid plexus failed to exhibit convincing biochemical markers that would attest in favor of a functional cholinergic innervation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1990

Muscarinic receptor subtype mediating vasodilation feline middle cerebral artery exhibits M3 pharmacology.

The nature of the muscarinic receptor subtype mediating the endothelium-dependent relaxation of the cat middle cerebral artery was investigated in vitro by recording the smooth muscle isometric tension of precontracted arterial segments. Relaxation induced by several agonists (acetylcholine (ACh), acetyl-beta-methylcholine, oxotremorine, carbachol and McN-A-343) was recorded. The ability of selective (pirenzepine, dicyclomine, adiphenine, AF-DX 116, methoctramine, gallamine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and hexahydro-sila-difenidol (HHSiD] and non-selective antagonists (atropine, scopolamine and quinuclidinyl benzilate (QNB] to block the relaxation induced by ACh was also estimated. The weak activity of the poorly selective M1 muscarinic receptor as together with the intermediate affinity of pirenzepine and adiphenine tend to exclude the M1 muscarinic receptor as the primary mediator of the cholinergic relaxation. The low affinity of AF-DX 116 and methoctramine further suggested that the cerebrovascular muscarinic receptor does not correspond to the M2 cardiac subtype. In contrast, 4-DAMP and HHSiD potently inhibited the ACh-induced relaxation with affinities similar to those reported at the M3 glandular receptor. We conclude that a similar to the pharmacological M3 muscarinic receptor subtype is responsible for the cholinergic relaxation of the cat middle cerebral artery.

Acetylcholine↗