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5-hydroxytryptamine (5-HT) is a key regulator of muscle contraction in parasitic flatworms. In Schistosoma mansoni, the myoexcitatory action of 5-HT is effected through activation of a serotonergic GPCR (Sm.5HTRL), prioritizing pharmacological characterization of this target for anthelmintic drug discovery. Here, we have examined the effects of several aporphine alkaloids on the signaling activity of a heterologously expressed Sm.5HTRL construct using a cAMP biosensor assay. Four structurally related natural products – nuciferine, D-glaucine, boldine and bulbocapnine – were demonstrated to block Sm.5HTRL evoked cAMP generation with the potency of GPCR blockade correlating well with the ability of each drug to inhibit contractility of schistosomule larvae. Nuciferine was also effective at inhibiting both basal and 5-HT evoked motility of adult schistosomes. These data advance our understanding of structure-affinity relationships at Sm.5HTRL, and demonstrate the effectiveness of Sm.5HTRL antagonists as hypomotility-evoking drugs across different parasite life cycle stages., Graphical abstract, Highlights • Application of a cAMP biosensor to probe properties of a schistosome serotonergic GPCR. • Several aporphine alkaloid derivatives act as 5-HT receptor antagonists. • Potency of GPCR blockade correlates well with inhibition of schistosomule contractility. • The natural product nuciferine acts as an effective inhibitor of adult worm motility.

The cytochrome P450 2C19 (CYP2C19) enzyme plays an important role in the metabolism of many commonly used drugs. Relatively little is known about CYP2C19 inhibitors, including compounds of natural origin, which could inhibit CYP2C19, potentially causing clinically relevant metabolism-based drug interactions. We evaluated a series (N = 49) of structurally related plant isoquinoline alkaloids for their abilities to interact with CYP2C19 enzyme using in vitro and in silico methods. We examined several common active alkaloids found in herbal products such as apomorphine, berberine, noscapine, and papaverine, as well as the previously identified mechanism-based inactivators bulbocapnine, canadine, and protopine. The IC50 values of the alkaloids ranged from 0.11 to 210 µM, and 42 of the alkaloids were confirmed to be time-dependent inhibitors of CYP2C19. Molecular docking and three-dimensional quantitative structure-activity relationship analysis revealed key interactions of the potent inhibitors with the enzyme active site. We constructed a comparative molecular field analysis model that was able to predict the inhibitory potency of a series of independent test molecules. This study revealed that many of these isoquinoline alkaloids do have the potential to cause clinically relevant drug interactions. These results highlight the need for studying more profoundly the potential interactions between drugs and herbal products. When further refined, in silico methods can be useful in the high-throughput prediction of P450 inhibitory potential of pharmaceutical compounds.

Cissampelos capensis, commonly known by the Afrikaans name “dawidjies” or “dawidjieswortel”, is the most important and best known medicinal plant of the family Menispermaceae used by the Khoisan and other rural people in the western region of South Africa. The main alkaloids in the leaves, stems and rhizomes were isolated and identified. Several of the main compounds were previously found in species of the related genus Antizoma and this similarity indicates that the two genera are closely related if not congeneric. Bulbocapnine (an aporphine alkaloid), dicentrine (an aporphine alkaloid) and salutaridine (a morphinane alkaloid) were the main alkaloids in the leaves, while bulbocapnine, cissacapine, cycleanine and insularine (the last three are bisbenzyltetrahydro-isoquinoline alkaloids) are the major compounds in the stems. The rhizome contains mostly bisbenzyltetrahydroisoquinoline alkaloids, with 12-O-methylcurine, cissacapine and cycleanine as the main ones. Alkaloids appear to be quite variable within different plant parts and different provenances, as confirmed by the difference in alkaloid patterns between coastal and inland forms of Cissampelos capensis (the morphinane alkaloid salutaridine, for example, is the major leaf alkaloid along the coast but is practically absent from the inland form of the species). The variety of alkaloids identified may contribute to the medicinal value of this species. The data on alkaloidal variation in the species has potential value and practical applications in chemotaxonomy, toxicology and pharmacognosy.

Although many studies have been carried out on the dopaminergic system, little is known about the dopaminergic system in the ovary. The present study was undertaken to investigate the role of the dopamine (DA) system in ovarian function especially in steroidogenesis using rat ovaries. Ovarian cells from PMS-treated rats were incubated for 1 hour with or without DA or other drugs. DA, norepinephrine (NE) and isoproterenol (Iso) increased the levels of progesterone (P4) and cAMP in the media. D1 agonists (SKF38393, SKF82526-J, CY208-243) increased P4 secretion, whereas bromocriptine (D2 agonists) did not show any effect on the P4 level in the media. The effect of NE and Iso on P4 and cAMP levels was inhibited by propranolol (Pro; beta-blocker), while the increase of P4 and cAMP levels caused by DA or D1 agonists was suppressed by bulbocapnine (Bul; D1 antagonists). Propranolol (beta-blocker) or domperidone (D2 antagonists) did not affect the levels of P4 and cAMP. The presence of dopamine D1 receptor in the PMS-treated rat ovary was revealed by a kinetic study. The maximal number of binding sites (Bmax) of ovarian D1 receptor was 1.33fmol/mg tissue and the Kd value was 0.357nM. These results suggest that the DA system may physiologically play a role in the steroidogenesis in the ovary through D1 receptor.

Little is known about the dopamine system in the ovary. The present study has been undertaken to investigate the effect of dopamine (DA) on the ovarian steroidogenic enzymes of pregnant mare serum gonadotropin (PMSG)-treated immature rats. Ovarian cells from PMSG-treated rats were cultured for 1-5 hours with or without DA, D1 agonists or bulbocapnine (Bul)(D1 antagonist). Progesterone (P) and estradiol (E2) in the media were assayed by specific RIAs. The enzyme activities were assayed by adding radioactive substrates in the media before incubation. DA and D1 agonists increased P in the media which was caused by the increment of 3 beta -hydroxysteroid dehydrogenase (3 beta -HSD) activity because cholesterol side chain cleavage enzyme (CSCC) activity showed no significant change. The stimulating effects of DA and DA agonists on P and 3 beta -HSD activity were inhibited by Bul. DA showed no effect on 17 alpha-hydroxylase activity. DA decreased 17.20 lyase activity, but this decrement was probably a non specific effect. DA alone did not affect the E2 level in the media and aromatase activity. The present results suggest that DA mainly stimulated 3 beta -HSD activity of the PMSG-treated rat ovary which regulated P synthesis.

We examined the possibility that dopamine may play a role in nerve-mediated "off" responses of esophageal body (EB) and relaxations of lower esophageal spincter (LES) smooth muscle. The effects of dopamine, epinine, and dopamine antagonists on EB and LES smooth muscle were studied on these responses. Dopamine and epinine caused a dose-related fall in basal LES muscle tension and in amplitude of EB muscle "off" responses. Threshold dose for both was about 10(-7) M, and maximal dose was about 10(-4) M. At high concentrations, they also caused repetitive transient contractions of both LES and EB muscle after the period of inhibition. These effects were antagonized by haloperidol, 10(-5) M, and bulbocapnine, 10(-5) M, but were not influenced by propranolol, 10(-5) M, nor by phenoxybenzamine, 10(-5) M. Neither haloperidol nor bulbocapnine influenced responses to electrical field stimulation. Tetrodotoxin 10(-7) M abolished the responses to electrical field stimulation but did not antagonize the effects of dopamine and epinine. EB and LES smooth muscle contain a dopamine receptor. It is unlikely that dopamine is involved in responses to electrical field stimulation.

Opossum duodenum was cut into 2 mm X 1.5 cm strips. The strips cut along the oral-caudal axis were called longitudinal strips, while those cut 90 degrees to that axis were called circular strips. Dopamine produced contractions in both muscle layers. ED 50 for the longitudinal and circular muscle was 2.8 X 10(-6) M and 1.9 X 10(-5) M, respectively. The longitudinal muscle was 6.5 times more sensitive to dopamine than the circular muscle. The dopamine-produced contractions were completely blocked by phenoxybenzamine, 10(-4) M, and phentolamine, 10(-5) M. Haloperidol, 10(-5) M, or bulbocapnine, 10(-5), completely or partially blocked the contractions produced by dopamine in only one-third of the strips. Tetrodotoxin, propranolol, atropine, and curare did not affect the dopamine-produced contractions. These studies suggest that dopamine mainly stimulates alpha-excitatory adrenergic receptors in opossum duodenal muscle and that the longitudinal muscle is more sensitive to dopamine. However, we cannot exclude the existence of excitatory dopamine receptors in the opossum duodenal smooth muscle.

Field stimulation of the parasympathetic nerves supplying the rabbit isolated rectococcygeus muscle produced individual twitch responses that were inhibited by dopamine (1 x 10(-8)-1 x 10(-5)M). The twitch-inhibitory effect of dopamine was reversed by haloperidol or sulpiride (1 x 10(-8)-1 x 10(-5)M for either) but not by phentolamine or yohimbine (up to 1 x 10(-4)M). Haloperidol and sulpiride were potent, specific, competitive antagonists of the twitch-inhibitory effect of dopamine; their pA2 values were 8.39 and 7.75, respectively. In contrast, cis alpha-flupenthixol, fluphenazine, bulbocapnine and thioridazine were weak or inactive against dopamine. Concentrations of dopamine that inhibited the twitch response to parasympathetic nerve stimulation had little or no effect on contractions elicited by carbachol or by direct muscle stimulation after abolition of neuronal conduction by tetrodotoxin. Thus, the effects of dopamine on responses elicited by parasympathetic nerve stimulation seem to be exerted at a prejunctional level rather than directly on the smooth muscle. The twitch-inhibitory effect of dopamine was mimicked by epinine, N,N-diethyldopamine, N,N-di-n-propyldopamine, 5,6-ADTN, N,N-di-n-propyl 5,6-ADTN, 6,7-ADTN, apomorphine and Sandoz 27-403. Sulpiride reversed the effects of all these agonists. N,N-di-n-propyl-6,7-ADTN and SK & F 82526 also inhibited the twitch response but their effects were not reversed by sulpiride. SK & F 38393 and DPI had little effect on the twitch response. The pharmacological characteristics of the presynaptic dopamine receptors in the rabbit rectococcygeus muscle show that they resemble those in the cat heart and rabbit ear artery in some respects but there are differences that suggest that the presynaptic dopamine receptors in the rabbit rectococcygeus muscle constitute a specific subgroup of receptors.

1 Noradrenaline and clonidine were applied by microiontophoresis to single neurones in the cerebral cortex and medullary reticular formation of anaesthetized rats.2 Of a total of 247 neurones studied, 79% of medullary units and 60% of cortical units responded in the same manner to both noradrenaline and clonidine. The usual response was a depression of neuronal firing rate.3 It proved possible to antagonize some responses to both substances by the microiontophoresis of bulbocapnine, whilst leaving unaffected similar responses to 5-hydroxytryptamine.4 On 13% of the cells, clonidine produced an increase of firing rate. This effect could not be attributed to a post-synaptic antagonism of tonically released endogenous noradrenaline, but may indicate a presynaptic action of clonidine, reducing noradrenaline release.5 These observations are thought to support the idea that clonidine may have an agonist action on noradrenaline receptors in the brain.

Time courses of the behavioural and biochemical effects of a cataleptogenic dose (50mgkg−1 i.p.) of bulbocapnine have been studied in the rat. Catalepsy ensues immediately after administration of the drug and lasts 1 h. Concomitant with the onset of catalepsy there is a rise in HVA and DOPAC concentrations in whole and discrete parts of the brain (striatum, limbic system). Dopamine content does not change in whole brain but it decreases in the striatum and increases in the cortex and hippocampus. No significant effects on NA, 5-HT and 5 HIAA concentrations were observed.

It is a well-known fact that both morphologically and functionally the extrapyramidal system is extremely complex. In these studies of the system I have introduced pharmacological methods in an endeavor to understand. and elucidate its functions from the pharmacological point of view. For this purpose, I have picked out the so-called “extrapyramidal poisons” that produce a certain syndrome by acting on the extrapyramidal system. In this way, I have been able to clarify that bulbocapnine, mescaline, harmine and harmaline are toxic substances of that particular nature. Since among these drugs harmine and bulbocapnine manifest the most characteristic actions and properties, I have thought it worthwhile to investigate with particular care the extrapyramidal actions and the “point of attack” on the central nerves of these substances in experiments on various animals. The results of these experiments will be given in a recapitulated form.

Prof. Hara has previously suggested on the two distinguished divisions in the extrapyramidal system, namely the bulbocapnine-reacting system and the harmine-reacting system. In his idea some experiments were carried out in order to explain the significance of striatum for the development of action of extrapyramidal poisons. The corpus striatum of a mouse was divided into six portions by means of the high frequency electrocautery. In determining the effect of harmine and bulbocapnine under these conditions, it was proved that the action of harmine was remarkably decreased when the anterior portion of striatum was injured, and that the injury of the posterior portion exerted a great reduction of action of bulbocapnine.

Since all of the so-called “major tranquilizers” have the ability to induce catatonic states in experimental animals, this characteristic pharmacological property is commonly being used as a clue to detecting new compounds in screening tests. However, not all compounds that produce the catalepsy in animals are “tranquilizers”. One remarkable example is bulbocapnine, an alkaloid isolated from Corydalis cava; it was first reported by Peters (1) that a catatonic state in mammals followed bulbocapnine treatment. Although it has been suggested that bulbocapnine catalepsy could serve as an experimental model for schizophrenia or Parkinson's disease, little is known of its effects on the neural components of the motor system. In earlier experiments we found that bulbocapnine selectively depressed the spinal monosynaptic reflex response in the rat with little effect on the polysynaptic neuronal systems (2). These observations have subsequently been confirmed by Willis et al. (3, 4) in the cat. The present report describes a further investigation of the actions of bulbocapnine on spinal reflex systems. The rat was employed in the present experiments so that the electrophysiological data could be accurately compared with the numerous behavioral observations in this species.

Sympathomimetic effects have been studied on the isolated vas deferens of the rat, in the isolated jejunum of the rabbit and on the cardiovascular system of the cat. (–)-Noradrenaline (1-R configuration), (+)-noradrenaline (1-S configuration) and dopamine as well as a number of homologues were used as agonists. The adrenergic blocking drugs piperoxan, phentolamine, yohimbine, aceperone and the tranquillising drugs chlorpromazine, (–)-mepromazine, haloperidol, droperidol and spiramide as well as bulbocapnine were used as antagonists. The results obtained with both agonists and antagonists provide evidence that the structural requirements for drugs to react with and to activate α-receptors in the vas deferens and the rabbit intestine are different; epinine and dopamine have an identical mechanism of action.

The phytochemical investigation of the alkaloid-rich fraction obtained from the leaves of Guatteria pogonopus Mart. (Annonaceae) allowed the isolation and identification for the first time in this species of: (+)-nornuciferine (1), a mixture of 1 and (+)-anonaine (2), (+)-isocorydine (3), (+)-nuciferine (4), (+)-roemerine (5), (-)-tetrahydropseudocolumbamine (6), a mixture of 6, liriodenine (9) and lysicamine (10), a mixture of 1,2,9-trimethoxy-10-hydroxyaporphine (7) and bulbocapnine (8), 9, 10, and (+)-N-methyllindicarpine (11). Compounds 6, 7, 8, and 11 have not been previously reported in the family Annonaceae. Furthermore, the formerly synthetic 1,2,9-trimethoxyaporfin-10-ol (7) is described for the first time as a natural aporphine alkaloid herein. The chemical structures were established by 1D and 2D NMR as well as in comparison with data previously reported in the literature. The cytotoxic activity of the alkaloids was evaluated against tumor (B16-F10, HepG2, HL-60, and K562) and non-tumor (PBMC) cell lines. Alkaloid 1 presented significant activity against HepG2 cell lines with IC50 of 9.60 µmol L-1 while the mixture of 6, 9 and 10 displayed strong cytotoxic activity against HL-60 and K562 cell lines with IC50 values of 3.41 an 8.50 µmol L-1, respectively.

Antagonist activity of bulbocapnine on DA1 versus DA2 dopamine receptors was studied simultaneously in a dog under pentobarbitone anaesthesia and without phenoxy-benzamine pretreatment. Fenoldopam (SK&F 82526) injected into the renal artery was the DA1 agonist and dipropyl dopamine or piribedil injected into the femoral artery were the DA2 agonists. Both at 0.5 and 1 mg kg−1 doses intravenous bulbocapnine caused nearly equal inhibition of DA1 and DA2 dopamine receptor-mediated responses. Our results show that under these experimental conditions bulbocapnine is not a selective DA1 dopamine antagonist.

An in vivo model is presented for studying the vasodilator effect of dopamine on the gastric vascular bed of the anesthetized rabbit. Dopamine was injected into the common hepatic artery simultaneously measuring the blood flow through the left gastric artery at constant perfusion pressure. The vasoconstrictor response to dopamine was blocked by pretreatment with phenoxybenzamine (0.5 mg/kg i.a.). Dopamine dilated the gastric vascular bed dose-dependently with an average ED50 of 15 nmol. Apomorphine caused vasodilation with an ED50 of 38.8 nmol; the maximum response was significantly lower than dopamine. (-)Isoproterenol was ineffective. cis-Flupentixol (3.5 and 7.0 mumol/kg i.v.) and (+)butaclamol (3.0 mumol/kg i.v.), but not trans-flupentixol (7.0 mumol/kg i.v.) and (-)butaclamol (6.0 mumol/kg i.v.) shifted the dopamine dose-response curve to the right in a parallel fashion, indicating competitive antagonism. Haloperidol, bulbocapnine, and sulpiride also acted as competitive inhibitors of dopamine-induced vasodilation. The antagonists inhibited the dopamine-induced vasodilation in the following order of potency: (+)butaclamol greater than cis-flupentixol greater than haloperidol greater than bulbocapnine greater than sulpiride. The results suggest that the vascular dopamine receptors in the stomach of the rabbit resemble the DA1-receptors in the mesenteric vascular bed of dogs.

The objective of the present experiments was to determine the specific receptor subtype through which dopamine (DA) receptor agonists relax the lower esophageal sphincter in vitro. Opossum lower esophageal sphincter smooth muscle strips were placed in oxygenated Krebs' solution containing propranolol and cocaine. The tissues were placed at a tension that gave maximum relaxation to electrical field stimulation and were then pretreated with phenoxybenzamine. The effects of DA, and the DA receptor agonists epinine and apomorphine were determined. In addition, agonist responses were studied in the presence of the selective DA2 receptor antagonist domperidone, a mixed DA1/DA2 receptor antagonist metoclopramide, and the selective DA1 receptor antagonists bulbocapnine and SK&F 83566. The DA agonists relaxed the smooth muscle strips in the following order of potency: DA greater than epinine greater than apomorphine. Domperidone did not antagonize DA- or apomorphine-induced relaxation. Metoclopramide failed to alter DA-induced relaxation. Bulbocapnine and SK&F 83566 significantly inhibited the relaxation induced by DA. These data indicate that DA-induced lower esophageal sphincter relaxation in vitro is mediated by DA1 receptors.

On rat isolated vas deferens, apomorphine was found to be l/5th as active as dopamine. Reserpine or cocaine pretreatment failed to reduce the response to apomorphine. Low concentrations of apomorphine which do not cause contraction of the tissue, antagonize the effects of dopamine. The effects of dopamine were antagonized equally by phentolamine or apomorphine. On rabbit aortic strips 4 × 10−4M apomorphine repeated at 45 min intervals induces pronounced tachyphylaxis. During the tachyphylactic period dopamine was not inhibited to a significantly greater extent than was phenylephrine. Bulbocapnine tested against dopamine and phenylephrine yielded identical pA2 values of 6. Only at 10−5M was bulbocapnine observed to produce a preferentially greater blockade of dopamine. Dopamine or isoprenaline-induced relaxations of aortic strips were not blocked by bulbocapnine. Dopamine and 3 times 10−6M bulbocapnine increased the chronotropic effects in atria. Apomorphine and higher doses of bulbocapnine produced rate decelerating effects. On atria low doses of apomorphine were equally effective in reducing the effects of dopamine or histamine. These results are discussed in light of the proposal that the effects of apomorphine and bulbocapnine involve dopamine receptor interactions. In all three tissues there was no clear cut evidence of specific dopamine receptors.