Kenneth L. Dretchen

Sequence specific detection of DNA using nicking endonuclease signal amplification (NESA).

We have developed a new method for identifying specific single- or double-stranded DNA sequences called nicking endonuclease signal amplification (NESA). A probe and target DNA anneal to create a restriction site that is recognized by a strand-specific endonuclease that cleaves the probe into two pieces leaving the target DNA intact. The target DNA can then act as a template for fresh probe and the process of hybridization, cleavage and dissociation repeats. Laser-induced fluorescence coupled with capillary electrophoresis was used to measure the probe cleavage products. The reaction is rapid; full cleavage of probe occurs within one minute under ideal conditions. The reaction is specific since it requires complete complementarity between the oligonucleotide and the template at the restriction site and sufficient complementarity overall to allow hybridization. We show that both Bacillus subtilis and B. anthracis genomic DNA can be detected and specifically differentiated from DNA of other Bacillus species. When combined with multiple displacement amplification, detection of a single copy target from less than 30 cfu is possible. This method should be applicable whenever there is a requirement to detect a specific DNA sequence. Other applications include SNP analysis and genotyping. The reaction is inherently simple to multiplex and is amenable to automation.

Effect of electric and chemical stimulation of the raphe obscurus on phrenic nerve activity in the cat

The effect of electrical and chemical (L-glutamate) stimulation of the raphe obscurus on phrenic nerve activity was examined in the cat. Phrenic nerve activity was recorded from a C5 nerve root in anesthetized, paralyzed and artificially ventilated cats. Neural discharge was quantitated by integrating the phrenic nerve activity. The respiratory frequency was determined from the integrated nerve signal. Focal electrical stimulation (18-144 microA; 5-40 Hz; 100 microseconds pulse duration) resulted in significant (P less than 0.05) increases in both integrated phrenic nerve (IPN) amplitude and respiratory frequency. These changes were dependent upon current intensity and frequency of stimulation. The largest increases in IPN amplitude and respiratory frequency were 47 +/- 17% and 146 +/- 8%, respectively. To insure that the changes in integrated phrenic nerve activity (IPNA) were the result of stimulation of cell bodies and not axons of passage, L-glutamate (100, 200 nmol) was microinjected (100 nl) into the raphe obscurus. Significant (P less than 0.05) dose-related changes occurred in integrated phrenic nerve amplitude with an increase of 44 +/- 13% at 100 nmol and 80 +/- 13% at 200 nmol L-glutamate. No significant increase in respiratory frequency was observed with L-glutamate microinjection. The results suggest that the raphe obscurus may be involved in respiratory control.

Azathioprine: Effects on Neuromuscular Transmission

The neuromuscular effects of azathioprine were examined in the in-vivo cat soleus muscle preparation. In concentrations ranging from 10 to 1,000 μg/kg. administered intra-arterially, the agent caused motor axons to fire repetitively and produced a dose-related increase in the force of contraction. The drug reversed neuromuscular blockade produced by d-tubocurarine and potentiated the neuromuscular blockade produced by succinylcholine. The effects of theophylline, a phosphodiesterase inhibitor, on neuromuscular transmission were identical to those produced by azathioprine. Using an in-ritro assay preparation, azathioprine was found to produce 50 per cent inhibition (IC30) of phosphodiesterase at a concentration of 2 × 10−3M. In the same preparation, theophylline had an IC30 of 1 × 10−4 M. Neither agent in concentrations to 10−2 M affected cholinesterase activity measured in ritro. It is concluded that the effects of azathioprine on neuromuscular transmission are due to inhibition of phosphodiesterase in the motor nerve terminal.

Sympathetic nervous system mediated cardiovascular effects of cocaine are primarily due to a peripheral site of action of the drug

Currently, augmentation of sympathetic nervous system function produced by cocaine is thought to be due primarily to stimulation of sympathetic centers in the brain (central effect) and to inhibition of catecholamine uptake into postganglionic sympathetic nerve terminals (peripheral effect). In this review of our work, we present the following evidence that cocaine-induced changes in cardiovascular function, particularly those that peak within 1 to 5 min after an i.v. bolus injection of the drug, are due to a peripheral effect of the drug: (1) In both dogs and cats, cocaine potentiates the tachycardiac effect of neurally-released and injected norepinephrine (NE). The time course of action and dosage range of cocaine that produces potentiation follows that which increases blood pressure (BP), heart rate (HR), rate-pressure product and coronary vasoconstriction. (2) Cocaine given in i.v. doses that increase BP in decerebrate cats (0.25-1.0 mg/kg) has no significant effect on directly monitored spontaneous cardiac sympathetic nerve activity (SNA). In fact, higher doses of cocaine (2-4 mg/kg, i.v.) consistently inhibit preganglionic cardiac and splanchnic nerve activity. (3) Cocaine (0.1-1.0 mg) administered directly into the blood supply of the hindbrain via the vertebral artery produces no increase in BP, HR or SNA in cats; instead, decreases in BP and sympathetic activity occur. The same dose (1 mg), injected i.v., does not depress BP or SNA. In addition, cocaine injected into the forebrain via the carotid artery or into the cerebral ventricles (0.1-1.0 mg) has very little effect on BP. Our results indicate that there is no significant excitatory effect of cocaine on CNS sympathetic centers, and that the sympathomimetic effects of cocaine on the cardiovascular system are likely to be mediated at peripheral sites.

Experimental Studies on the Neurocardiovascular Effects of Urapidil

SummaryThe major purpose of our study was to determine whether urapidil acts in the central nervous system (CNS) to lower arterial blood pressure. Once demonstrating a CNS antihypertensive action of urapidil we further set out to determine: (1) the relative role of a CNS antihypertensive action to the total antihypertensive effect of urapidil; (2) the brain site of action for the antihypertensive effect of urapidil; and, (3) the receptor mechanism whereby urapidil acts in the CNS to lower arterial blood pressure. Studies were conducted in chloralose-anaesthetised cats, and arterial blood pressure and heart rate were monitored. Drugs were administered intravenously (IV), into the cerebral ventricles (ICV), topically by application to the ventral surface of the medulla and by microinjection into specific nuclei. Receptor binding studies were also conducted using rat cerebral cortex homogenates. We found that injection of urapidil into the fourth ventricle decreased arterial pressure. Local application of urapidil to the ventral medullary surface also decreased arterial blood pressure. Microinjection of urapidil into one of the nuclei associated with the ventral surface of the medulla, the rostral part of the nucleus reticularis lateralis (rLRN), produced a similar degree of antihypertensive effect. The effect of urapidil was not altered by α1-receptor blockade. Instead, the urapidil effect resembled that produced by drugs that stimulate serotonin (5-hydroxytryptamine)-1A receptors (B695-40 and 8-OH-DPAT). Furthermore, urapidil was found to have the highest potency for binding to serotonin-1A receptor sites (as compared to α1- and α2receptor sites). Urapidil administered IV was shown to lower arterial blood pressure in part by blocking peripheral α1-adrenoceptors but also, in high doses, by acting in the CNS to decrease central sympathetic outflow. These data indicate that urapidil is a unique drug, possessing both peripheral and CNS actions which contribute to its antihypertensive effect. Urapidil may also be unique in that its central action may involve activation of serotonin-1A receptors.

Hypotensive effect of urapidil: CNS site and relative contribution.

Summary: The purposes of our study were to determine the contribution of the CNS to the hypotensive effect of urapidil in the cat and the specific brain site of action of this agent. For the first purpose, urapidil was studied on preganglionic sympathetic nerve activity, arterial pressure, and heart rate. Three systemic bolus doses of urapidil were administered (0.22, 0.44, and 1.3 mg/kg). All three doses lowered arterial pressure, and the highest dose produced a significant decrease in sympathetic nerve discharge in five of six animals studied. The lower two doses had no significant effect on sympathetic activity, and none of the doses altered heart rate. These results suggest that a high i.v. dose of urapidil is required to evoke hypotension by an action in the central nervous system (CNS). For the second purpose, urapidil was applied bilaterally to the intermediate area of the ventral surface of the medulla in doses of 25 and 50 µg. These doses caused decreases in arterial pressure of −6.1 ± 2.2 (p < 0.05) and −21.0 ± 5.9 (p<0.05) mm Hg, respectively, but no change in heart rate. In addition, respiratory stimulation also occurred with the higher dose as respiratory minute volume increased by 81 ± 14 ml/min (p < 0.05). The highest dose of urapidil had no effect on arterial pressure when applied to other chemosensitive areas of the ventral surface of the brain. Comparative studies with prazosin (10 µg applied bilaterally to the intermediate area) indicated no hypotensive effect of this α1-adrenoceptor blocking agent. These results suggest that the central hypotensive effect of urapidil occurs at the intermediate area and does not involve blockade of α1-adrenoceptors.

Protection by phenytoin and calcium channel blocking agents against the toxicity of diisopropylfluorophosphate

Pretreatment of male Swiss-Webster mice with phenytoin, 25 mg/kg, verapamil, 25 to 3.0 mg/kg, nifedipine, 0.05 to 0.1 mg/kg, nitrendipine, 0.1 mg/kg, and nimodipine, 1 to 2.5 mg/kg, elevated the LD50 of diisopropylfluorophosphate (DFP) to a significant degree. In addition, these agents enhanced the protection that can be obtained from atropine and 2-pralidoxime. The protective effects of phenytoin cannot be attributed to an anticonvulsant action, per se, since carbamazepine, phenobarbital, and diphenylbarbituric acid in anticonvulsant doses did not influence DFP lethality. The mechanism of action of phenytoin and the other effective calcium channel blockers in providing protection over and above that achieved with atropine and 2-pralidoxime appears to be due to a protective action of the former agents on central respiratory centers and peripheral nicotinic sites and may involve the movement of calcium into excitable membranes.

Dantrolene sodium: Effects of smooth muscle

The effects of dantrolene sodium on smooth muscle were evaluated in vitro using the guinea pig ileum and the guinea pig vas deferens preparations. Dantrolene irreversibly decreased the strength of the responses of the ileum to acetylcholine, histamine, potassium chloride and electrical stimulation. Doubling the concentration of calcium in the bathing media reduced the effect of dantrolene on all agonists. Dantrolene irreversibly decreased the response of the vas deferens to epinephrine, norepinephrine, acetylcholine and electrical stimulation. Doubling the concentration of calcium in the bathing media reduced the effect of dantrolene on all agonists. Dantrolene seems to depress smooth muscle as it does skeletal muscle. The mechanism of action probably involves the reduction of calcium flux into the muscles.

Characterization of the train-of-four response in fast and slow muscles: effect of d-tubocurarine, pancuronium, and vecuronium.

The in vivo cat soleus and gastrocnemius muscles were used to compare isometric contraction strength and the train-of-four (T4) response (2 Hz for 2 s) of two muscle types (fast and slow) during onset of competitive neuromuscular blockade in order to determine the extent of the correlation between twitch depression and T4 fade. Prior to drug administration the muscles that were studied differed significantly in that the T4 ratio was 1.0 in the gastrocnemius and only 0.87 in the soleus. Three competetive neuromuscular-blocking agents were compared: d-Tubocurarine, pancuronium, and vecuronium. d-Tubocurarine was found to produce a close correlation between the degrees of twitch strength depression and T4 for both muscles. However, these muscles demonstrated significantly different ED50 values (105 μg/kg for gastrocnemius, 150 μg/kg for soleus). Pancuronium also produced a similar relationship between twitch strength depression and T4 decrement for each muscle. In this case, however, there was little difference in their Ed50 values for twitch depression (11.5 μg/kg for gastrocnemius, 13 μg/kg for soleus). The effects of vecuronium were quite different from the other two muscle relaxants. Although vecuronium produced a comparable correlation between twitch tension and T4 fade in fast muscle, no such relationship was found to exist in slow muscle. Even when the twitch strength was blocked to 18% of control, the soleus T4 response was depressed to only 75% of control. These results higlight major differences among competitive neuromuscular-blocking agents and suggest multiple sites of action.

Short communicationProtection by phenytoin and calcium channel blocking agents against the toxicity of diisopropylfluorophosphate

Pretreatment of male Swiss-Webster mice with phenytoin, 25 mg/kg, verapamil, 25 to 3.0 mg/kg, nifedipine, 0.05 to 0.1 mg/kg, nitrendipine, 0.1 mg/kg, and nimodipine, 1 to 2.5 mg/kg, elevated the LD50 of diisopropylfluorophosphate (DFP) to a significant degree. In addition, these agents enhanced the protection that can be obtained from atropine and 2-pralidoxime. The protective effects of phenytoin cannot be attributed to an anticonvulsant action, per se, since carbamazepine, phenobarbital, and diphenylbarbituric acid in anticonvulsant doses did not influence DFP lethality. The mechanism of action of phenytoin and the other effective calcium channel blockers in providing protection over and above that achieved with atropine and 2-pralidoxime appears to be due to a protective action of the former agents on central respiratory centers and peripheral nicotinic sites and may involve the movement of calcium into excitable membranes.