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To investigate the underlying ionic mechanism of the late plateau phase of the action potential in rabbit atrium the whole-cell patch-clamp technique with intracellular perfusion was used. We recorded the inward current during repolarizations following a brief 2 ms depolarizing pulse to +40 mV from a holding potential of between -70 and -80 mV. The development of this current coincides with the onset of the late plateau phase of the action potential. Peak activation of the current occurs about 10 ms from the beginning of the depolarizing pulse, and it decays spontaneously with a slow timecourse. Its voltage dependency from -40 mV to +40 mV shows very steep activation (-40 to -20 mV) and shows almost the same maximum magnitude between -10 mV and +40 mV. This behaviour is quite different from that of the calcium current. The inward current and the late plateau phase of the action potential were both abolished by the application of 5 mM EGTA, 1 microM ryanodine and by reducing the Na+ gradient. The fully activated current-voltage relation of the inward current was plotted as the difference current before and after treatment with Ryanodine, Diltiazem, 20 mM Na+ inside or 30% Na+ outside and shows an exponential voltage dependence with the largest magnitude of the current occurring at negative potentials. The current-voltage (I-V) curve was well fitted by the Na-Ca exchange equation, i = A exp (-(1 - r)EF/RT). The results suggest that the inward current contributes to the generation of the late plateau phase of the rabbit atrial action potential, and is activated by intracellular calcium released from the sarcoplasmic reticulum. Sarcoplasmic reticulum calcium release appears to be triggered both by the membrane voltage and by the calcium current. It is concluded that the inward current is generated by Na-Ca exchange.

1. The ability of five different tissues of the rat to bind homologous and heterologous (human) low density lipoprotein (LDL) was compared. 2. Human LDL was as effective as rat LDL in displacing rat [125I]LDL from the plasma membrane fraction of rat skeletal muscle. 3. Membranes from rat liver appeared to have no affinity for homologous LDL, while human LDL was slightly more effective in displacing labeled rat LDL from this tissue. 4. A similar pattern of displacement was observed in membranes derived from rat heart. 5. In contrast, membranes from both the aorta and adrenal glands of the rat displayed a significantly greater affinity for binding LDL of homologous origin. 6. These results suggest that lipoprotein receptors analogous to those described for cultured human cells may exist in the rat and that individual tissues exhibit distinctly different affinities for binding LDL of both homologous and human origin.

The 3 major lipoprotein fractions of human serum were investigated for their ability to inhibit the haemagglutination of 24 different togaviruses including rubella virus and arboviruses from 5 antigenic groups. All viruses were inhibited by the 3 fractions and this was most evident with low and very low density lipoproteins; inhibitory activity appeared to be associated with the lipid part of the lipoproteins. When artificial dispersions of the lipids cholesterol, cholesteryl ester and triglyceride were emulsified with egg lecithin three categories of inhibitory activity were observed which were independent of virus antigenic grouping -- category 1, no activity; category 2, activity confined to cholesterol and category 3, activity for cholesterol and to a lesser extent the other lipids.

Neuroblastoma x glioma cells NG108-15 were cultured in lipid-free medium supplemented with fatty acids of various chain length and unsaturation. Binding of 3H-labelled [DAla2]-[DLeu5]-enkephalin by membranes of cells grown in saturation fatty acids of different chain length was not significantly different from that of the control On the other hand, a proportional decrease of binding capacity with no change in residual receptor affinity was noticed when cells were cultured in medium containing fatty acids of increasing unsaturation. This decrease was time dependent and reached a maximum at about 48 h. Binding of [3H]dihydromorphine and [3H]naloxone was similarly affected. In contrast, when membranes of cells grown in normal medium were preincubated up to 3 h with unsaturated fatty acid and tested for opioid binding, no significant reduction was observed. Examination of the fatty acid composition of phospholipid from cells grown in linolenate indicated that a significant alteration of the acyl composition has occurred. To evaluate the underlying cause of this type of inhibition, the effect of linolenic acid on cell growth and protein synthesis was examined. When cells were cultured in 100 microM of this fatty acid, both growth and protein synthesis were retarded by 28% and 19%, respectively. Since opiate receptors are proteineous in nature, a reduction of protein synthesis may partially account for the loss of opioid binding activity. On the other hand, an increase of membrane fluidity is known to affect a number of cellular functions, including ligand-receptor recognition. Whether this can offer a satisfactory explanation for our observations remains to be established.

The effects of dietary habit, body weight, physical activity and a familial record of coronary heart disease on serum lipid and lipoprotein levels in Chinese were examined. Increased dietary preference for animal meat products was associated with increased serum cholesterol and beta-lipoprotein levels. Increased carbohydrate intake and overweight were associated with increased serum triglyceride and pre-beta-lipoprotein levels. A familial record of coronary heart disease was associated with increased serum total lipid and pre-beta-lipoprotein levels. Based on these results, the factors which influence the serum lipid and lipoprotein concentrations in Chinese are probably similar to those in other ethnic groups.

We have constructed a cDNA library in lamda gt11 using mRNA isolated from the pituitary glands of the grass carp (Ctenopharyngodon idellus). Based on the published sequence of the rainbow trout growth hormone cDNA, we synthesized two oligonucleotide probes. One of these hybridized strongly with a specific mRNA fragment from the grass carp pituitary. Using this probe, we have isolated six positive clones carrying an insert of approximately 1.2 Kb. By restriction enzyme digestion, all the clones were determined to be identical. Sequence determination on one of them indicated that it has an open reading frame coding for 210 amino acids. Both the nucleotide and translated amino acid sequence are highly homologous to those of the salmon growth hormone and the common carp. A putative signal peptide consisting of hydrophobic amino acids can be identified at the 5' end of the sequence. A polyadenylation signal, ATTAAA, was also present 12 base upstream from the poly A tail.

Forty-two heroin addicts and 31 normal persons were examined for the effect of acupuncture and electrical stimulation (AES) on plasma ACTH, cortisol and cyclic-AMP levels. Both ACTH and cortisol levels were reduced significantly in the addicts after treatment whereas no such significant reduction was observed in the normals. Plasma cyclic-AMP level was not affected in either group. Taken together, results from the present study suggest that the mechanism of AES in the treatment of addiction may have a neuroendocrinological basis. This hypothesis is particularly attractive in view of the isolation of opiate-like peptides from the brain.

The suitability of using dodecylamine-gel for removing the serum non-antibody-like inhibitors of haemagglutination by rubella was studied. Compared with kaolin and MnCl2/heparin treatment this new procedure appears to have a higher specificity since it removes the non-antibody-like inhibitors from serum without affecting the immunoglobulin level significantly. The potential application of this procedure in routine serological analysis for rubella virus infection is discussed.

The postnatal changes in the levels of radioimmunoassayable enkephalin and beta-endorphin, as well as the densities of [3H]methionine-enkephalin and [3H]naloxone binding sites in rat cerebellum, brainstem and whole forebrain were determined. The opiate peptides and the opiate binding sites reached their highest levels at the first week postpartum in the cerebellum, at the second week in the brainstem and at the third week in the whole forebrain. This finding is in line with the developmental profiles of other well-established neuronal pathways which also showed a caudal-to-rostral sequence of development. Moreover, there was a close relationship between the elevation and decline in the amounts of opiate binding sites and in the levels of opiate peptides in each brain region. These observations are consistent with other evidence which suggests that enkephalin and beta-endorphin are functioning as neurotransmitters or neuromodulators in the central nervous system.

The level of met-enkephalin in the cerebrospinal fluid of 18 schizophrenic and 18 non-mental patients were determined by radioimmunoassay. The concentration of this peptide in the schizophrenic patients ranged from 12 to 39 ng/l with a mean of 22.2 ng/l. The mean and range of the non-mental patients were 28 ng/l and 16 to 41 ng/l, respectively. The difference between the mean enkephalin level of the two groups was statistically significant by the t-test. However, this difference must be interpreted with caution since the data obtained from the non-mental patients cannot be regarded as normal. No significant correlation was observed between the severity of schizophrenic symptoms and cerebrospinal fluid met-enkephalin level.

The effectiveness of dynorphin-(1-13) in suppressing heroin withdrawal was studied. At a dose of 60 micrograms/kg body weight, dynorphin-(1-13) was effective in suppressing withdrawal symptoms. When saline was administered a strong placebo effect was noticed that, however, did not influence the outcome of the results. Only three out of the twelve patients receiving dynorphin-(1-13) experienced some mild side-effects such as feeling warm, dizziness and precordial formication.

Seven cases of chronic pain were treated by intrathecal administration of 30 micrograms of beta-endorphin and dynorphin-(1-13). Compared with saline, both peptides were able to suppress pain for periods up to 4.5 and 7 hours on the average, respectively. No significant side reactions were noticed during the entire investigation.

Thirteen patients with pain from various causes were treated by electroacupuncture for 30 min. Cerebrospinal fluid (CSF) was obtained before and after treatment. Opioid-like substances in the CSF were fractionated by high pressure liquid chromatography and assayed by competitive receptor binding using a mu-specific radioligand, [D-ala2, MePhe4, gly-ol5]-enkephalin (DAGO). Opioid activity, associated with a fraction, eluted at 18-20% acetonitrile, consistently showed an increase in level after acupuncture. Two other fractions eluted at larger concentrations of acetonitrile also increased significantly after acupuncture; however the increase was not consistently observed in every patient. Measurements of beta-endorphin and dynorphin by radioimmunoassay indicated that 80 and 60% of the patients, respectively, had a higher level of these peptides after acupuncture. The nature of the opioid activity, eluted at 18-20% acetonitrile is unknown; however a small amount of it could be found in various parts of the brain of rat.

Dynorphin-(1-13) and -(1-10) were administered by intrathecal injection into six terminal cancer patients at doses of 7.5, 15, 30 and 60 micrograms. Compared with saline, both analogues of dynorphin were effective in suppressing pain. The duration of relief at doses of 15 micrograms and above was more than 4 hours on the average for both peptides. However, no proportional increase in response was observed when the dose applied was doubled. This lack of response might have been due to the development of tolerance.

Tetramethylpyrazine, a drug originally isolated from the rhizome of Ligusticum walliichi, has been used routinely in China for the treatment of stroke and angina pectoris. We evaluated this drug by testing its effectiveness in increasing the survival rate in a stroke model using Mongolian gerbils. Our results indicate that tetramethylpyrazine can increase survival rate only if it is administered before the induction of cerebral ischemia. Since we administered the drug intraperitoneally, it is possible that pretreatment was necessary to increase its effective concentration in the blood. Receptor binding studies indicated that tetramethylpyrazine was inactive against a variety of pharmacologically active receptors.

beta-Endorphin-like immunoreactivity in whole brains of Sprague-Dawley rat fetuses of different gestational ages was measured by radioimmunoassay and found to increase throughout the gestational period studied. The immunoreactivity in various brain parts (forebrain, midbrain, hindbrain, hypothalamus and pituitary) of late prenatal, early postnatal, young mature and retired breeder rats was also determined. In all the brain parts studied, a maximum in the content and concentration of beta-endorphin-like immunoreactivity was attained when the rats were about 3-4 months old.

The serum concentrations of ACTH, cortisol, aldosterone, thyroxine, cyclic AMP and cholesterol were compared between normal and heroin-addicted subjects. Significantly lower ACTH, cyclic AMP and cholesterol levels were observed to be associated with the heroin addicts, but their plasma thyroxine level was significantly elevated. The possible physiopsychological effect resulting from these changes is discussed.

The present study was undertaken to evaluate if plasma or CSF beta-endorphin level can be induced to rise during the treatment of heroin addiction by electroacupuncture. Based on the examination of 30 addicts, we obtained no evidence indicating an increase of beta-endorphin level in either the plasma or the CSF after 30 min of acupuncture. In spite of this, the majority of the addicts experienced a reduction of withdrawal symptoms during treatment. Since electroacupuncture may only induce a highly localized secretion of beta-endorphin in the brain, our results cannot unequivocally exclude the possibility that this peptide is involved in mediating the action of acupuncture.

The effect of electro-acupuncture in the treatment of naloxone precipitated morphine withdrawal in mice was studied. Thirty minutes of acupuncture was enough to suppress withdrawal behaviors by more than 60%. Concomitant to the decrease of withdrawal behavior, there was a significant increase of brain beta-endorphin level in the acupunctured animals. Plasma beta-endorphin level of the treated and untreated group was not significantly different.

The mixture of human platelets and prostaglandin (PG) endoperoxide analogs (U46619 and U44069) at threshold concentrations caused a marked enhanced vasocontraction of rabbit aortae and bovine coronary arteries. THe configuration of the mixture-induced contraction was a sustained and tonic form. On the other hand, the thromboxane A2-like substance which was generated during the incubation of arachidonic acid with human platelets caused a transient and phasic contraction, and the amplitude of this contraction was much less than that of the mixture. Even after exposure to high temperature for various time periods, the mixture was still able to cause the enhanced vasocontraction. However, this mixture lost the activity of vasocontraction after an incubation in strong acid (pH 1) or alkali (pH 12) for 10 min. Serotonin and alpha adrenoceptor antagonists had no effect on the mixture-induced contraction. When tissues were incubated in Ca-free medium for a few minutes, the mixture failed to cause vasocontraction, whereas the contractile responses to histamine, norepinephrine and serotonin were not affected. When tissues were pretreated with imidazole (10(-5) M), which is a thromboxane synthetase inhibitor, the vasocontration induced by the mixture was potentiated rather than inhibited, whereas the thromboxane A2-like substance-induced contraction was inhibited. When either the tissue was pretreated with or platelets were incubated with indomethacin, the mixture failed to cause the enhanced vasocontraction. The new synthetic arginine derivatives (T1189 and T1233) nearly abolished the vasocontractile response to the mixture. However, both derivatives failed to inhibit the vasocontractions of PGE2 and PGF2 alpha, arachidonic acid and KCl. These results suggest the possibility that during the incubation of endoperoxides with human platelets a potent vasocontractile substance could be generated and such a substance does not resemble thromboxane A2 or any of the PGs tested.

Human serum lipoproteins were purified by ultracentrifuging and their concentrations adjusted as required to be within the normal male/female serum range for all assays. The activities in inhibition of hemagglutination (HAI) for Japanese encephalitis virus were--low density lipoprotein (LDL) greater than very low density lipoprotein (VLDL) greater than high density lipoprotein (HDL). Heating (56 degrees C/30 minutes) caused the LDL titer to fall and freeze-thawing (20 degrees C/room temperature) the VLDL titer to rise slightly, possibly as a result of alteration in lipoprotein structure. The additon of lipoprotein depleted serum appeared to dampen these effects and there was no nett change in titer when it was added to a lipoprotein mixture. Similarly, unfractionated normal serum showed no significant change in titer after these treatments. The lipoproteins lacked significant virus neutralizing (VN) activity and this remained so in spite of fluctuations in HAI titer after heating and freeze-thawing.

The role of serum lipoproteins as non-antibody-like inhibitors of haemagglutination for rubella virus was investigated. Dissociation of lipoproteins into their respective lipid and protein components significantly reduced their inhibitory titre. This reduction was more prominent with the protein component. When mixtures of pure lipids were tested for their haemagglutination inhibitory activities, no inhibition was observed. Sonication of the three major lipoprotein fractions significantly increased the inhibitory activities of low density lipoproteins (LDL) and very low density lipoproteins (VLDL) but not high density lipoproteins (HDL). Succinylation, acetylation, and methylation of the lipoproteins did not appear to affect their inhibitory titre. On the other hand, phospholipase A treatment significantly increased the inhibitory properties of all lipoproteins fractions. These results are discussed in terms of the possible mechanisms by which the lipoproteins interact with the rubella haemagglutinins.

The role of apo-low density lipoprotein in the regulation of low density lipoprotein receptor synthesis was studied. Fibroblasts preincubated in the presence of a cholesterol-free low density lipoprotein preparation showed an increase of 50% in receptor activity. In contrast, when cells were preincubated with a similar amount of normal low density lipoproteins a 2- to 3-fold decrease of receptor activity was observed. The increase of receptor activity induced by delipidated LDL was probably due to an increase of synthesis rather than an unmasking effect since treatment with puromycin essentially abrogated the activation. The ability of the partially delipidated low density lipoproteins to elicit receptor synthesis could not be accounted for by a depletion of cellular cholesterol because a similar preparation of delipidated high density lipoproteins was unable to induce receptor synthesis but was able to reduce cellular cholesterol to the same extent. Treatment with chloroquine, a lyzosome inhibitor, blocked the ability of the delipidated low density lipoprotein to activate receptor synthesis. This suggests that degraded fragments rather than the intact delipidated lipoprotein are necessary to trigger the cell to synthesize more receptors.

The effectiveness of beta-endorphin, dynorphin-(1-13), dynorphin-(1-10) amide, alpha-neoendorphin and [D-Ala2,D-Leu5]enkephalin in suppressing withdrawal in heroin addicts was compared in this study. Groups of six patients were stabilized overnight in the hospital and were treated with either saline or peptide when withdrawal symptoms began to appear the following morning. Withdrawal was scored before and after treatment by the patient himself and an independent observer. Peptides were administered in a bolus dose of 60 micrograms/kg body weight. The patient, the observer and the physician who administered the injection were all blind to the nature of the compound given. All treatments, including those with saline, produced an overall reduction of withdrawal score. However, by statistical analysis, only treatments with beta-endorphin, [D-Ala2,D-Leu5]enkephalin and dynorphin-(1-13) were effective in producing a significant decrease of withdrawal symptoms. The length of relief brought about by the different peptides varied from less than an hour to a maximum of 5 h in one case. The average period of relief brought about by beta-endorphin, dynorphin-(1-13) and [D-Ala2,D-Leu5]enkephalin was 44, 46 and 60 min, respectively. Of the five peptides administered [D-Ala2,D-Leu5]enkephalin produced the largest number of side-effects.

1. Two arginine derivatives that were developed as thrombin inhibitors (TI-189 and TI-233) selectively inhibited the 5-hydroxytryptamine (5-HT)-induced contraction of rabbit aortic strips in a competitive manner. The PA2 values of TI-189 and TI-233 were 5.24 +/- 0.21 and 6.23 +/- 0.32 respectively. 2. Even at 10(-4) M they had no inhibitory effect on the contractile response to noradrenaline (NA), histamine, prostaglandin E2 (PGE2), PGF2 alpha, arachidonic acid or potassium in rabbit aortic strips. 3. In dog basilar and coronary arterial strips and also in rat fundus, both agents inhibited the 5-HT response in a non-competitive manner. 4. At 10(-5) M, TI-233 but not TI-189 antagonized effects of NA and KCl in the dog basilar and coronary arteries. 5. These arginine derivatives decreased the contractile responses induced by a prostaglandin endoperoxide analogue (U-44069) in rabbit aorta and in dog basilar and coronary arteries but there was no evidence for competitive antagonism. 6. These results indicate that the arginine derivatives are competitive antagonists selective for 5-HT receptors in rabbit aorta.

Lymphocytes isolated from morphine addicted mice show a number of impaired immunological functions. They include Con A stimulated mitogenesis, NK cell activity, plaque forming cell activity and delayed-type of hypersensitivity. In addition, the met-enkephalin and beta-endorphin level in the lymph nodes of addicted mice were also depressed.

Plasma ACTH, cortisol, and cyclic-AMP levels of eleven heroin addicts were dertermined before and after treatment with a fast detoxification procedure using acupuncture and electrical stimulation (AES) together with the administration of limited doses of naloxone. At the end of the treatment period, the average plasma ACTH, cortisol, and cyclic-AMP level rose 130,83, and 24 percent, respectively. In view of the appearance of mild withdrawal signs during this method of treatment, the observed increases in ACTH and cortisol levels probably reflect the inability of AES to suppress withdrawal symptoms induced by naloxone completely. The mechanism underlying this new method of treatment is discussed in relation to AES's ability to stimulate the secretion of endorphins.

The effect of naloxone on CSF [Met5]enkephalin level and on the suppression of psychotic symptoms associated with schizophrenia was studied. Seven patients were treated with naloxone at a dose of 0.4 mg/day for seven days and six were treated with saline for the same period. Of the seven patients receiving naloxone, three showed signs of improvement. In the saline group, only one case of obvious improvement was observed. The mean CSF [Met5]enkephalin level before and after treatment in both the naloxone and saline group did not change significantly. However, there was a highly significant correlation (r = -0.73, P less than 0.01) between the increase of [Met5]enkephalin level and the decrease in psychotic symptoms.

Eight female heroin-addicted subjects were treated over a 10-day period by acupuncture and electrical stimulation (AES). Their plasma ACTH, cortisol and c-AMP levels were reduced after initial AES treatment. The reduction of c-AMP was significant the most of the three. The suppression of these compounds may be associated with the temporary relief of withdrawl symptoms on and after each AES treatment in the first three days. On the third day, ACTH, cortisol and c-AMP levels did not show significant changes after AES. This was presumably due to the disappearance of withdrawal symptoms and possibly to the stabilizing effect of the treatment. Both the ACTH and c-AMP levels were generally lower at the end of the treatment period. Results of the present investigation suggest that plasma c-AMP may be the best parameter by which to gauge the response of heroin addicts to AES.

The intestinal contents of a dog infected with 49,732 M. yokogawai for 8 weeks were normal but in a 4-week-old infection the intestinal water content correlated roughly with the number of worms. Most worms were found 40 to 102 cm along the small intestine but the entire length was parasitized. The concentrations of Na+, K+ and Cl- decreased and the concentration of HCO3- and pH increased along the small intestine while the osmolarity remained roughly constant (324 to 340 mOsm/kg).

A protein of molecular weight approximately 12,000 which binds long-chain fatty acids and certain other lipids has been identified in cytosol of intestinal mucosa, liver, myocardium, adipose tissue, and kidney. Binding is noncovalent and is greater for unsaturated than for saturated and medium-chain fatty acids. This protein appears to be identical with the smaller of two previously described cytoplasmic anion-binding proteins. Binding of long-chain fatty acids by this protein is greater than that of other anions tested, including sulfobromophthalein, and does not depend on negative charge alone. The presence of this binding protein may explain previously observed differences in intestinal absorption among fatty acids, and the protein may participate in the utilization of long-chain fatty acids by many mammalian tissues.