Ileum preparation

Crude and three diethyl ether extracted, acetone treated, fractions were isolated from large-scale cultures of Gambierdiscus toxicus. Crude extracts at. 04 mg/ml inhibited the histamine contraction response in smooth muscle of the guinea pig ileum. Three semi-purified fractions at 5 ng/ml, effectively inhibited the guinea pig ileum preparation. Two of these fractions followed Michaelis-Menten kinetics for a competitive inhibition. The third fraction inhibited in a non-reversible manner. This study has established the presence of three lipid extracted toxins in toxicus, outlined a method for their assay in small quantities, and identified at least two of the effects of these toxic extracts in animals. 

Figure 3. Contractile response of guinea pig ileum preparation as a function of the log of the histamine concentration and the concentration of the toxin in the saline solution.

Reports of effects on mice observed for maitotoxin closely resemble the effects of ciguatoxin, with the exception of diarrhea, hypersalivation, and convulsions before death. The symptoms observed here for ESAP did not include hypersalivation and only occasionally was mild diarrhea observed. The vasodilation in the ears, ptosis of the eyelids and abdomen were observed in this study but not in other accounts. Crude extracts produced irreversible inhibition of the frog nerve muscle preparation, affecting first the synapse, secondly the nerve and lastly the muscle (23). At. 04 mg/ml the crude extract completely inhibited the isolated guinea pig ileum preparation. 

GT-3 was separated from GT-1 and GT-2 on silicic acid using 100% methanol. The separation of this fraction is the same as that used by Tachibana (22) and Yasumoto (13) for maitotoxin. Unlike GT-1 and GT-2, this fraction was found to be an irreversible inhibitor of the guinea pig ileum preparation. The time studies of GT-3 are important in that they characterize GT-3 as a very toxic fraction but one which is very slow in its action. 

Relationship of Fractions to Other Toxins. Our initial extractions of tHe toxic fractions are tHe same as that of Tachibana, ( ) and others. Thus, the isolation of GT-la and GT-lb correspond exactly to the initial steps in the isolation of ciguatoxin by Scheuer ( ). The isolation of GT-2b and GT-2c and their similar action on the ileum preparation, causes us to conclude that they are either a more polar form of GT-1 or a modification of GT-1 accomplished during the extraction procedures. GT-3, it appears, would most likely correspond to a carry over of a maitotoxin-1ike fraction from the initial ether-water separation. Without any chemical confirmation, however, these are only tentative identifications. Indeed, toxins extracted from Caribbean isolates of toxicus, could be quite different from those extracted from Pacific isolates. 

Use of the Ileum Preparations. With respect to the dose response curves and the conclusions that one may try to draw from these, we understand the arguments surrounding the classical receptor theory and that there are limits to both the precision and accuracy that one can expect for the technique. Nevertheless, we infer several things from our data. 

In summary, we have isolated three toxins from lipid extracts of tqxicus. We have demonstrated that the guinea pig ileum preparation is an effective assay for these toxins in nanogram quantities. All three fractions effectively inhibited the guinea pig ileum preparation in its response to histamine at nanogram concentrations. The GT-3 toxic fraction is quite different from the other two (GT-1, GT-2) in being very slow acting and nonreversible in nature. 

Menkveld, G.J., Timmerman, H., 1990. Inhibition of electrically-evoked contractions of guinea-pig ileum preparations mediated by the histamine H3 receptor. Eur. J. Pharmacol. 186, 343-347. 

In the GPI, /J-FNA produced a potent reversible agonist response, but upon incubation of the ileum preparation, it gave rise to an irreversible an-... 

Isovaltrate and valtrate (valepotriates) and valeronone, an essential oil component, isolated from Valeriana edulis ssp. procera Meyer (Valeriana "mexicana") caused suppression of rhythmic contractions in guinea pig ileum in vivo at a dose of 20 mg/kg administered intravenously via the jugular vein. The investigators also demonstrated that the same compounds as well as dihydrovaltrate isolated from the same valerian species produced relaxation of carbachol-stimulated guinea pig ileum preparations in vitro. They concluded that these compounds have a musculotropic action in concentrations from 10 5. to 10 4M(Hazelhoff et al., 1982). 

Many studies using the guinea-pig ileum preparation have shown that Ca + is able to antagonize the inhibitory effects of opiates on electrically induced contractions (31-34, 36,37)... 

Lis Balchin et al. [69] studied the action of S. sclarea oil on the rat isolated phrenic nerve diaphragm preparations and compared with activity on field- stimulated guinea-pig ileum preparations. The oil produced a contracture and inhibition on the skeletal muscle of the bi-phasic response to nerve stimulation, whilst only a contracture, with or without a decrease in response to field stimulation, was produced in smooth muscle. 

Fig- (t7). Log cumulative concentration-contraction curves for contraction induced by synthesized aminoacyl cholines in ileum preparations isolated from guinea pig. Contraction responses were expressed as % of the contraction induced by acetylcholine (ACh, 5.5 pM). The values represent means S.E.M. (n = 4-7). 

The guinea pig ileum assay appears to be able to distinguish three separate lipid soluble toxins obtained from G. toxicus cultures. These toxic fractions isolated from G. toxicus show inhibition of the guinea pig ileum preparations (Miller et al. 1984). 

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