Thermo-mechanical behaviors of thermoplastic starch derived from sugar palm tree (Arenga pinnata).

In recent years, increasing environmental concerns focused greater attention on the development of biodegradable materials. A thermoplastic starch derived from bioresources, sugar palm tree was successfully developed in the presence of biodegradable glycerol as a plasticizer. Sugar palm starch (SPS) was added with 15-40 w/w% of glycerol to prepare workable bioplastics and coded as SPS/G15, SPS/G20, SPS/G30 and SPS/G40. The samples were characterized for thermal properties, mechanical properties and moisture absorption on exposure to humidity were evaluated. Morphological studies through scanning electron microscopy (SEM) were used to explain the observed mechanical properties. Generally, the addition of glycerol decrease the transition temperature of plasticized SPS. The mechanical properties of plasticized SPS increase with the increasing of glycerol but up to 30 w/w%. Meanwhile, the water absorption of plasticized SPS decrease with increasing of glycerol.

3-Morpholinosyndnonimine inhibits 5-hydroxytryptamine-induced phosphorylation of nitric oxide synthase in endothelial cells.

5-Hydroxytryptamine (5-HT) is a vasoactive substance that is taken up by endothelial cells to activate endothelial nitrite oxide synthase (eNOS). The activation of eNOS results in the production of nitric oxide (NO), which is responsible for vasodilation of blood vessels. NO also interacts with superoxide anion (O2*-) to form peroxynitrite (ONOO-), a potent oxidant that has been shown to induce vascular endothelial dysfunction. We examined the ability of 3-morpholinosyndnonimine (SIN-1), an ONOO- generator, to inhibit 5-HT-induced phosphorylation of eNOS in cultured bovine aortic endothelial cells (BAECs). We observed that 5-HT phosphorylates Ser1179 eNOS in a time- and concentration-dependent manner. Maximum phosphorylation occurred at 30 sec using a concentration of 1.0 microM 5-HT. BAECs treated with SIN-1 (1-1000 microM) for 30 min showed no significant increase in eNOS phosphorylation. However, 5-HT-induced eNOS phosphorylation was inhibited in cells treated with various concentrations of SIN-1 for 30 min and stimulated with 5-HT. These data suggest that an increase in ONOO- as a result of an increase in the production of O2*-, may feedback to inhibit 5-HT-induced eNOS phosphorylation at Ser1179 and therefore, contribute to endothelial dysfunction associated with cardiovascular diseases.

5-hydroxytryptamine stimulates phosphorylation of p44/p42 mitogen-activated protein kinase activation in bovine aortic endothelial cell cultures.

5-Hydroxytryptamine (5-HT) is sequestered and released by endothelial cells, acts as an endothelial cell mitogen, promotes the release of nitric oxide (NO), and has been associated with the p44/p42 mitogen-activated protein kinase (MAPK) cascade. NO also acts as a cell mitogen and promotes signals that culminate in the phosphorylation of MAPK. The aim of this study was to test whether endothelial 5-HT receptors stimulate dual (tyrosyl- and threonyl-) phosphorylation of MAPK through a mitogen-activated protein kinase kinase-1 (MEK-1) and eNOS-dependent pathway in bovine aortic endothelial cells (BAECs). As shown by Western blot analysis, 5-HT and the 5-HT1B-selective agonist 5-nonyloxytryptamine (5-NOT) stimulate time- and concentration-dependent (0.001-10 microM) phosphorylation of MAPK in these cells. The agonist-stimulated phosphorylation of MAPK was blocked by the 5-HT1b-receptor antagonist isamoltane (0.01-10 p3M) and the MEK-1 inhibitor PD 098059 ([2-(2'-amino-3'-methoxy-phenyl)-oxanaphthalen-4-one]; 0.01-10 microM¿. The eNOS inhibitor L-N(omega)-iminoethyl-L-ornithine (L-NIO; 0.01-10 microM) failed to block the 1 microM 5-NOT-stimulated responses. Our findings suggest that the 5-HT receptors (specifically 5-HT1B) mediate signals to MEK-1 and subsequently to MAPK through an eNOS-independent pathway in BAECs.

5-hydroxytryptamine evokes endothelial nitric oxide synthase activation in bovine aortic endothelial cell cultures.

Activation of endothelial nitric oxide synthase (eNOS) results in the production of nitric oxide (NO) that mediates the vasorelaxing properties of endothelial cells. The goal of this project was to address the possibility that 5-hydroxytryptamine (5-HT) stimulates eNOS activity in bovine aortic endothelial cell (BAEC) cultures. Here, we tested the hypothesis that 5-HT receptors mediate eNOS activation by measuring agonist-stimulated [3H]L-citrulline ([3H]L-Cit) formation in BAEC cultures. We found that 5-HT stimulated the conversion of [3H]L-arginine ([3H]L-Arg) to [3H]L-Cit, indicating eNOS activation. The high affinity 5-HT1B receptor agonist, 5-nonyloxytryptamine (5-NOT)-stimulated [3H]L-Cit turnover responses were concentration-(0.01 nM to 100 microM) and time-dependent. Maximal responses were observed within 10 min following agonist exposures. These responses were effectively blocked by the 5-HT1B receptor antagonist, isamoltane, the 5-HT1B/5-HT2 receptor antagonist, methiothepin, and the eNOS selective antagonists (0.01-10 microM): L-Nomega -monomethyl-L-arginine (L-NMMA) and L-N omega-iminoethyl-L-ornithine (L-NIO). Pretreatment of BAEC cultures with pertussis toxin (PTX; 1-100 ng/ml) for 16 hr resulted in significant inhibition of the agonist-stimulated eNOS activity, indicating the involvement of Gi proteins. These findings lend evidence of a 5-HT1B receptor/eNOS pathway, accounting in part for the activation of eNOS by 5-HT. Further investigation is needed to determine the role of other vascular 5-HT receptors in the stimulation of eNOS activity.

Cholinergic vasodilatory effect of an imidazoindole on the nephrosclerosis of hypertensive rats.

Pyridino [1,2-a] imidazo [5,4-b] indole (Compound 1) has been reported previously as an antihypertensive agent. Parenteral introductions of Compound 1 in spontaneously hypertensive rats (SHR) and in normotensive dogs have been found to reduce systolic blood pressure (SBP) in those animal models. Later studies have shown cholinomimetic effects of Compound 1 in animal tissue preparations and in live animals. Our experiments showed that daily oral administration of Compound 1 minimized the development of hypertensive nephrosclerosis and prevented premature death in male SHR. Compound 1 also potentiated cholinergic activity in isolated rabbit hearts. It is suggested that cholinergic vasodilation by Compound 1 is responsible for the hypotensive effect and prevention of nephrosclerosis in SHR.

Potentiation of cholinergic activity with pyridino[1,2-a]imidazo[5,4-b]indole: in vitro studies.

1. Effects of a novel imidazoindole derivative on cholinergic function were studied in isolated tissue preparations. 2. The compound demonstrated a dose-dependent (10(-11)-10(-9) potentiation (20-60%) of acetylcholine induced tension in guinea pig ileal tissue. 3. Increases in the size of end-plate potentials and nerve evoked muscle twitches were observed in frog nerve-skeletal muscle preparations. 4. Cholinesterase activity was not inhibited. 5. The results suggest that the compound has actions at the post-synaptic muscarinic receptor complex in smooth muscle and causes pre-synaptic increases in ACh release at the neuromuscular junction.

A study of the novel synthetic analog (+/-)-depentylperhydrohistrionicotoxin on the nicotinic receptor-ion channel complex.

(+/-)-Depentylperhydrohistrionicotoxin [(+/-)-depentyl-H12-HTX] is a synthetic analog of perhydrohistrionicotoxin (H12-HTX) that lacks the 5-carbon side chain. Recent studies with N-benzylazaspiro analogs of histrionicotoxin (HTX) in which both side chains are removed revealed that this alteration restricted the action of the compounds on the acetylcholine receptor-ionic channel complex (AChR) to an open channel blockade. Thus, an important question was raised as to the role of the side chains in affecting the interaction of these inhibitors with the AChR. In addition, the effect of (+/-)-depentyl-H12-HTX on membrane excitability was investigated. (+/-)-Depentyl-H12-HTX blocked the indirectly elicited twitch without affecting the directly elicited twitch. It decreased the amplitude and rate of rise and prolonged the falling phase of the action potential and blocked delayed rectification suggestive of blockade of sodium and potassium conductances. However, its effects on sodium and potassium conductances were less marked than those of HTX. It decreased the peak amplitude of the end-plate currents (EPCs) and accelerated the decay time constant of EPCs (tau EPC) in a concentration-dependent manner. The analog also induced voltage- and time-dependent nonlinearity in the current-voltage relationship of EPCs. Despite marked shortening of tau EPC, the decay phase of the EPC remained a single exponential function of time. Single channel conductance was unaffected by the analog, but the single channel lifetime was shortened. The voltage- and time-dependent effects of the analog that occurred without prior activation of AChR suggest reaction with the ionic channel in its closed conformation.

Interaction of analogs of histrionicotoxin with the acetylcholine receptor ionic channel complex and membrane excitability.

The effects of the four N-benzylazaspiro analogs of histrionicotoxin, which are without the two side-chains typical of histrionicotoxin, were studied on the ionic channels of electrically excitable membrane and the nicotinic acetylcholine receptors in frog sartorius muscles. Each analog reversibly blocked the indirectly elicited twitch and potentiated the directly elicited twitch in a concentration-dependent manner. The analogs decreased the amplitude and rate of rise and prolonged the falling phase of the directly elicited action potential and blocked delayed rectification suggesting blockade of sodium and potassium conductances. All of the analogs caused a concentration- and voltage-dependent depression of the peak end-plate current amplitude and induced nonlinearity but no hysteresis or time dependency in the current-voltage relationship. The marked shortening of the time constant of end-plate current decay produced by the analogs was concentration-dependent. The relationship between the time constant of end-plate current decay and membrane potential remained a single exponential function of time despite the marked shortening of the decay phase and loss of voltage dependence. The effect of the analogs on miniature end-plate current was identical to that on end-plate current. Single channel conductance was unaffected by the analogs, but the single channel lifetime was shortened. The marked shortening of the time constant of the end-plate current decay and single channel lifetime plus linear relationship between reciprocal of the time constant of decay and analog concentrations strongly suggest that the analogs interact with the ionic channels of the nicotinic acetylcholine receptor in their open conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

The nature of the interactions of pyridostigmine with the nicotinic acetylcholine receptor-ionic channel complex. I. Agonist, desensitizing, and binding properties.

The actions of pyridostigmine (Pyr), an anticholinesterase agent, were studied on the acetylcholine (ACh) receptor-ion channel complex and on the electrically excitable membrane of the frog cutaneous pectoris and sartorius muscles and the chronically denervated soleus muscle of the rat. Pyr at concentrations of 0.2-0.4 mM potentiated the indirect evoked muscle twitch and at concentrations greater than or equal to 0.8 mM depressed the indirect twitch with an IC50 of about 2 mM. Twitch depression produced by Pyr was reversed slowly, and after a 60-min wash only 59% of the control muscle twitch had returned. Pyr did not affect either the membrane potential or the muscle action potential. Pyr had several effects at the neuromuscular junction of the frog and rat. It decreased the peak amplitude of the end-plate current (EPC) in a voltage- and concentration-dependent manner. In contrast to diisopropylfluorophosphate, which depresses the EPC amplitude and induces a double exponential decay of the EPC and miniature end-plate current (MEPC), Pyr produced a marked prolongation of the time constants of EPC and MEPC decay while maintaining a single exponential decay. The decrease caused by Pyr of indirect twitch tension, EPC amplitude, and ACh sensitivity indicates mechanisms which limit the number and/or properties of conducting channels. The drug decreased channel conductance and prolonged channel lifetime as revealed by Fourier analysis of ACh-induced end-plate current fluctuations. An altered form of the conducting species induced by Pyr appears to be responsible for either the apparent agonist-induced depolarization or its ability to increase the affinity of ACh for its recognition site. Pyr was also found to inhibit the binding of ACh and alpha-bungarotoxin to receptor-rich membrane from the electric organ of Torpedo nobiliana, and to have a higher affinity for the receptor than for the ion channel binding sites. These actions are distinct from acetylcholinesterase inhibition caused by the agent. Strong evidence suggests that the direct influences of the agent on neuromuscular transmission involve at least three distinct, although possibly interacting, mechanisms: (a) a weak agonist action, (b) the formation of desensitized receptor-complex intermediates, and (c) the alteration of the conductance properties of active channels.

Interaction of bicyclo-octane analogs of amantadine with ionic channels of the nicotinic acetylcholine receptor and electrically excitable membrane.

A series of bicyclo-octane analogs of amantadine was investigated for their actions on ionic channels of electrically excitable membrane and of nicotinic acetylcholine receptors in frog sartorius muscles. Amantadine and three of its amine-substituted bicyclo-octane analogs (ID 11, 33 and 36) blocked neuromuscular transmission in a concentration-dependent manner. The dipropylaminomethyl analog (ID 36) also potentiated the directly elicited twitch concurrent with a prolongation of the directly elicited action potential; there was no block of delayed rectification. The peak amplitude of the end-plate current at -90 mV was reduced in a nearly equipotent manner by amantadine and ID 11, 33 and 36, but these three analogs were more potent than amantadine in shortening the time constant of end-plate current decay. The pattern of nonlinearity in the current/voltage relationship and area of negative slope conductance coupled with the depression and shortening of the end-plate current suggest that these analogs block neuromuscular transmission by interacting with the ionic channel of the acetylcholine receptor. Because carboxy-substituted analogs were ineffective and amine-substituted ones were effective, it appears that the site(s) which controls the opening and closing of the ionic channel require(s) a drug to penetrate a lipid barrier to a hydrophilic site in the membrane.

The ionic channel of the nicotinic acetylcholine receptor is unable to differentiate between the optical antipodes of perhydrohistrionicotoxin.

The enantiomers of perhydrohistrionicotoxin were studied in their effects on endplate currents recorded at the junctional region of sartorius muscles of Rana pipiens. The two optical antipodes progressively decreased the peak amplitude of the endplate currents and were indistinguishable from each other at all times. The enantiomers shortened equally the time constants for endplate current decay, but did not alter their voltage sensitivities. Although perhydrohistrionicotoxin contains 4 chiral centers, complete steric inversion does not alter its effects on the acetylcholine receptor-ion channel complex. By contrast the recognition site of the AcChR is extremely sensitive to any change in the chirality of agonists.

Meproadifen reaction with the ionic channel of the acetylcholine receptor: potentiation of agonist-induced desensitization at the frog neuromuscular junction.

The actions of the nicotinic noncompetitive antagonist meproadifen on both the acetylcholine (ACh) receptor-ion channel complex and electrically excitable membrane were examined in frog sciatic-nerve sartorius muscle preparations. Meproadifen (10-25 microM) blocked the nerve-evoked twitch without affecting the directly evoked twitch, the threshold, overshoot, amplitude, rate of rise, or falling phase of the directly elicited action potential in muscle. This suggests that this agent, at the concentrations that affect the nicotinic receptor, had negligible effect on the excitable membrane. In addition, the drug did not affect either the quantal content or quantal size of the end-plate potential. Meproadifen caused a voltage- and time-dependent decrease in the peak amplitude of the end-plate current (EPC) without significantly shortening the time constant of EPC decay. The voltage- and time-dependent effects of meproadifen were more pronounced at more negative potentials, as evidenced by hysteresis loops and nonlinearity in the current-voltage relationship of the EPC. Both hysteresis and nonlinearity in the current-voltage relationship of the EPC were eliminated when brief conditioning pulses were used for stepwise changes of membrane potentials. The decay time constant of the EPC in the presence of meproadifen remained an exponential function of time. Meproadifen blocked iontophoretically elicited EPCs but did not affect single-channel lifetime, conductance, or the decay time constant of the miniature EPC. Thus, the blockade was more marked on iontophoretically elicited EPCs than on miniature EPCs. Meproadifen also caused desensitization of both the junctional and extrajunctional ACh receptors, but, more important, meproadifen accelerated steady-state desensitization by several-fold (compared with the agonist). The marked depression of peak EPC amplitude and miniature EPC, its high affinity for the binding sites in the presence of the agonist, and acceleration of agonist-induced desensitization suggest that meproadifen interacts with the ACh-bound but nonconducting state of the ACh receptor-ion channel complex. Therefore, it appears that meproadifen interacts with the closed ionic channel of the ACh receptor in its resting and activated but nonconducting states, and only slightly affects the open conformation of the ionic channel.

(+/-)-2-Depentylperhydrohistrionicotoxin: a new probe for a regulatory site on the nicotinic acetylcholine receptor-channel.

(+/-)-2-Depentylperhydrohistrionicotoxin (4), several of its analogues, and N- and O-substituted derivatives were prepared and tested for their effects on the neuromuscular transmission of the frog sartorius muscle. Compound 4, its N-methyl derivative 5, the O-acetyl derivative 9, and the quaternary methiodides 19 and 20 blocked the indirectly elicited twitch. The oxidation of 4 and 5 to ketones 12 and 14 and their reduction to the epimeric alcohols 17 and 18 afforded materials with substantially reduced activity. N-Acetylation of 4 to 11 changed the course of the activity to a transient potentiation of muscle twitch. Both 4 and 5 were not very toxic to mice after subcutaneous administration. (+/-)-7-n-Butyl-1-azaspiro[5,5]undecan-8-one (12) epimerized readily at room temperature to afford the epimer 13, and preparation of the hydrochloride of its N-methylated derivative 14 was accompanied by a retro-Michael reaction, affording the 2-n-butyl-3-[4-(methylamino)butyl]cyclohexene-2-one (22). The strongly hydrogen-bonded alcohol 4 was analyzed as the hydrobromide by a single-crystal X-ray analysis, confirming its structure.

Structure-activity relationships of amantadine. I. Interaction of the N-alkyl analogues with the ionic channels of the nicotinic acetylcholine receptor and electrically excitable membrane.

In this study the effects of amantadine (1-adamantanamine) and its N-alkyl-substituted analogues [N-methyl- (NMA), N-ethyl- (NEA), N-propyl- (NPA), N-butyl- (NBA), and N,N-diethyl-amantidine (NNDEA)] were investigated on ionic channels of the electrically excitable membrane and of the nicotinic acetylcholine (ACh) receptors in frog sartorius muscles and on the binding of perhydrohistrionicotoxin (H12-HTX) to isolated membranes of the electric organ of the electric ray Torpedo. Amantadine and each analogue blocked the indirectly elicited twitch, but NPA, NBA, and NNDEA also potentiated the directly elicited twitch. The order of potency in inhibiting the indirect twitch was: NEA = NPA = NNDEA (10 microM) greater than NMA (15 microM) greater than NBA (40 microM) much greater than amantadine (130 microM). Neither amantadine nor its N-alkyl analogues affected miniature end-plate potential frequency or resting membrane potential but decreased miniature end-plate potential amplitude. Each compound prolonged the directly elicited action potential but did not alter delayed rectification. All of the compounds induced a concentration-dependent depression of the peak end-plate current (EPC) amplitude at negative membrane potentials and induced nonlinearity in the response at membrane potentials more negative than -40 mV. The order of potency in inhibiting the EPC (at -90 mV) was NNDEA (less than 0.5 microM) greater than NPA (less than 1.0 microM) greater than NBA (less than 2.0 microM) greater than NEA (19 microM) greater than NMA (42 microM) greater than amantadine (64 microM). Only NPA, NBA, and NNDEA depressed the peak EPC amplitude at positive membrane potentials as well. The shortening of the time constant of EPC decay by all compounds was concentration-dependent. At the higher concentrations examined, the slope of the relationship between the time constant of decay and membrane potential was reversed for all compounds. Only NPA induced a double-exponential decay of the EPC at positive membrane potentials. Neither amantadine nor its N-alkyl analogues inhibited the binding of [3H]ACh to its receptor in Torpedo electroplax but they inhibited the binding of [3H]H12-HTX binding to the ionic channel sites of the ACh receptor. The Ki for inhibition of [3H]H12-HTX binding was NEA = NNDEA (15 microM) greater than NMA (30 microM) greater than NPA = NBA (40 microM) greater than amantadine (60 microM). A gross correlation exists between their ability to block the indirect muscle twitch, miniature end-plate potential amplitude, peak EPC amplitude and the binding of [3H]H12-HTX. But, no correlation was found between these potencies and their antiviral activity. It is suggested that these compounds may interact with the ionic channel of the ACh receptor in its open and closed conformation.

Actions of the histrionicotoxins at the ion channel of the nicotinic acetylcholine receptor and at the voltage-sensitive ion channels of muscle membranes.

Various histrionicotoxins tested on frog nerve-muscle preparations showed a qualitative family resemblance to one another. They blocked the nerve-evoked muscle twitch and depressed both the peak amplitudes and the decay time constants of end-plate currents. During repetitive stimulation they progressively decreased the rate of rise and prolonged the falling phase of muscle action potentials, the latter resulting, at least in part, from blockade of voltage-sensitive potassium channels. These results indicated that the histrionicotoxins act at three membrane channels: the channel associated with the acetylcholine receptor, the sodium channel, and the potassium channel. Closer study of perhydrohistrionicotoxin suggested either two topographically distinct sites of action at the acetylcholine receptor-ion channel complex, or one site and two ion channel complex conformations. One site or conformation only alters the kinetics of channel closure. As these sites become saturated, the end-plate current decay time constant asymptotically approaches a limiting value. The other site or conformation prevents the channel from opening altogether. Further analysis indicated that the binding site for perhydrohistrionicotoxin that alters the kinetics of channel closure has an affinity constant of 0.1 microM-1 at -90 mV and that this affinity may be sensitive to the membrane potential. The lipid protein interface is a suggested site of histrionicotoxin action, common to the three channels studied here as well as to other intrinsic membrane proteins affected by histrionicotoxins.

[3H]Phencyclidine: a probe for the ionic channel of the nicotinic receptor.

To evaluate [3H]phencyclidine ([3H]PCP)as a probe for the ionic channel of the nicotinic receptor, the characteristics of its binding to electric organ membranes od Torpedo ocellata and its effects on frog sartorius muscle were studied. Similar to PCP, [3H]PCP depressed the peak amplitude of endplate current, caused nonlinearity in the voltage-current relationship at negative potentials, accelerated the decay time of the end-plate current, and shortened the channel lifetime. Thus, [3H]PCP interacted with the ionic channel of the nicotinic receptor, although there were a few differences between its effect and that of PCP. Binding of [3H]PCP to Torpedo membranes was to sites on the ionic channel of acetylcholine (AcCho) receptor because it was saturable, dependent upon protein concentration, and inhibited by drugs that interact with the ionic channel, and the initial rate of binding was potentiated by receptor agonists. Equilibrium binding of [3H]PCP to Torpedo membranes was with two affinities, but in the presence of AcCho, [3H]PCP binding was with a single affinity. The affinities of channel drugs obtained by inhibition of binding of [3H]PCP and [3H[perhydrohistrionicotoxin to Torpedo membranes were different, with correlation coefficients of 0.52 and 0.82 in the absence and presence of a receptor agonist, respectively; this suggests differences in their binding sites on the ionic channel of the AcCho receptor.