Pruritic and nociceptive sensations and dysesthesias from a spicule of cowhage.

Although the trichomes (spicules) of a pod of cowhage (Mucuna pruriens) are known to evoke a histamine-independent itch that is mediated by a cysteine protease, little is known of the itch and accompanying nociceptive sensations evoked by a single spicule and the enhanced itch and pain that can occur in the surrounding skin. The tip of a single spicule applied to the forearm of 45 subjects typically evoked 1) itch accompanied by nociceptive sensations (NS) of pricking/stinging and, to a lesser extent, burning, and 2) one or more areas of cutaneous dysesthesia characterized by hyperknesis (enhanced itch to pricking) with or without alloknesis (itch to stroking) and/or hyperalgesia (enhanced pricking pain). Itch could occur in the absence of NS or one or more dysesthesias but very rarely the reverse. The peak magnitude of sensation was positively correlated for itch and NS and increased (exhibited spatial summation) as the number of spicules was increased within a spatial extent of 6 cm but not 1 cm. The areas of dysesthesia did not exhibit spatial summation. We conclude that itch evoked by a punctate chemical stimulus can co-exist with NS and cutaneous dysesthesias as may occur in clinical pruritus. However, cowhage itch was not always accompanied by NS or dysesthesia nor was a momentary change in itch necessarily accompanied by a similar change in NS or vice versa. Thus there may be separate neural coding mechanisms for itch, nociceptive sensations, and each type of dysesthesia.

Valid across-group comparisons with labeled scales: the gLMS versus magnitude matching.

Labeled scales are commonly used for across-group comparisons. The labels consist of adjective/adverb intensity descriptors (e.g., "very strong"). The relative distances among descriptors are essentially constant but the absolute perceived intensities they denote vary with the domain to which they are applied (e.g., a "very strong" rose odor is weaker than a "very strong" headache), as if descriptors were printed on an elastic ruler that compresses or expands to fit the domain of interest. Variation in individual experience also causes the elastic ruler to compress or expand. Taste varies genetically: supertasters perceive the most intense tastes; nontasters, the weakest; and medium tasters, intermediate tastes. Taste intensity descriptors on conventional-labeled scales denote different absolute perceived intensities to the three groups making comparisons across the groups invalid. Magnitude matching provides valid comparisons by asking subjects to express tastes relative to a standard not related to taste (e.g., supertasters match tastes to louder sounds than do nontasters). Borrowing the logic of magnitude matching, we constructed a labeled scale using descriptors unrelated to taste. We reasoned that expressing tastes on a scale labeled in terms of all sensory experience might work. We generalized an existing scale, the Labeled Magnitude Scale (LMS), by placing the label "strongest imaginable sensation of any kind" at the top. One hundred subjects rated tastes and tones using the generalized LMS (gLMS) and magnitude matching. The two methods produced similar results suggesting that the gLMS is valid for taste comparisons across nontasters, medium tasters, and supertasters.

Spatial variation in sensitivity as a factor in measurements of spatial summation of warmth and cold.

Perception of cutaneous heating and cooling depends strongly on stimulus size. Although this dependence has been attributed solely to spatial summation, topographical variations in temperature sensitivity may also play a role. These variations, which differentially affect perception of small stimuli, may have led to overestimation of spatial summation. This possibility was investigated by measuring detection thresholds and perceived intensity for heating and cooling on the volar surface of the forearm using a multiple-thermode stimulus array. By keeping the array in place throughout each testing session we were able to measure threshold sensitivity and suprathreshold responsiveness at eight individual sites and for combinations of these sites having total stimulus areas of 0.64-5.12 cm2. When spatial summation was calculated in the traditional way by averaging the data for all stimuli of each size, the results agreed closely with previous estimates of summation for warmth and cold. When calculations were based instead on the most sensitive test site for each stimulus size, estimates of summation were reduced by about two-thirds. This outcome indicates that the spatial heterogeneity of thermal sensitivity likely contributed to estimates of spatial summation reported in earlier psychophysical studies. A schematic model of cutaneous thermoreception is presented that shows how neural summation and the density of innervation may combine to produce the psychophysical effects of increasing stimulus size (spatial enhancement).

Ibuprofen as a chemesthetic stimulus: evidence of a novel mechanism of throat irritation.

This paper reports a study of the oral and pharyngeal chemesthetic effects of the non-steroidal anti-inflammatory drug (NSAID) ibuprofen [2-(4-isobutylphenyl)propanoic acid], which pilot experiments had indicated produces an unusual sensory irritation of the throat. In experiment 1 subjects swallowed aqueous solutions of ibuprofen prepared with different buffering agents and gave ratings of irritation and taste in the mouth and throat. The results showed that ibuprofen irritates the throat much more than the mouth, and that its quality in the throat is characterized primarily as sting/prick, itch and tickle (often leading to cough). Based upon the results obtained with the different buffering agents, we hypothesized that the sting/prick/itch qualities of throat irritation were pH-dependent. Parametric manipulation of solution pH in experiment 2 confirmed this hypothesis. The same experiment revealed that, in contrast to other oral irritants (e.g. capsaicin and menthol), repeated stimulation caused neither sensitization nor desensitization of throat irritation. In the final experiment we found that ibuprofen's throat irritation could not be modulated by temperature, as it should be if stimulation occurred via capsaicin-sensitive receptors. We therefore conclude that ibuprofen has novel chemesthetic properties, which are not mediated by capsaicin-sensitive (vanilloid) receptors, and that a major component of the throat irritation it produces occurs via a pH-dependent receptor mechanism.

Inhibition and structure-activity studies of methionine hydroxamic acid derivatives with bacterial peptide deformylase.

The posttranslational deformylation of N-formyl-Met-polypeptides by the metalloenzyme, peptide deformylase, is essential for bacterial growth. Methionine hydroxamic acid derivatives were found to inhibit recombinant Escherichia coli peptide deformylase activity containing either zinc or cobalt. The binding of methionine hydroxamate and hydrazide inhibitors to cobalt-substituted deformylase caused spectral changes consistent with the formation of a pentacoordinate metal complex similar to that of actinonin, a psuedopeptide hydroxamate inhibitor. The spectral and kinetic data support the binding of these N-substituted L-methionine derivatives in a reverse orientation with respect to N-formyl-Met-peptide substrates within the active site. Based on this hypothesis a second generation of N-substituted methionyl hydroxamic acids were evaluated and found to possess greater inhibitory potency. These results may provide the basis for the design of more potent and selective deformylase inhibitors as potential antibacterial agents.

Substituted 2-aminopyridines as inhibitors of nitric oxide synthases.

A series of substituted 2-aminopyridines was prepared and evaluated as inhibitors of human nitric oxide synthases (NOS). 4,6-Disubstitution enhanced both potency and specificity for the inducible NOS with the most potent compound having an IC50 of 28 nM.

Measurement of sensory irritation of the skin.

The sensory reaction to contact with chemical irritants has not been extensively studied. This neglect has been caused partly by a lack of understanding of the physiological basis of subjective irritation, and partly by inadequate methods of perceptual measurement. This article begins with a brief overview of the neurophysiology of cutaneous chemoreception (chemesthesis), and continues with a discussion of numerous sensory, physical, and perceptual factors that can affect the sensitivity to irritants. Then, guidelines for conducting perceptual measurements of subjective irritation that take these factors into account are offered. An alternative approach to direct chemosensory measurement also is proposed that relies on the fact that many of the cutaneous receptors that are sensitive to chemicals also are sensitive to temperature and/or mechanical stimulation (pain or itch). It is suggested that thermal and mechanical sensitivity might prove useful as measures of the severity of acute sensory irritation and as indicators of subclinical sensitization by environmental irritants.

Menthol desensitization of capsaicin irritation. Evidence of a short-term anti-nociceptive effect.

Evidence is presented of a short-term antinociceptive effect of menthol that was discovered in the course of investigating menthol's potential to sensitize the mouth to capsaicin. Previous research had shown that treating the tongue with menthol 15 min before exposure to capsaicin could enhance the irritancy of capsaicin, and we wished to learn if this effect would increase as the time between exposure to menthol and capsaicin decreased. We found instead that when capsaicin followed menthol by only 3.5 min, or when it was presented in mixture with menthol for 2-3 min, sensory irritation was reduced rather than enhanced. We examined the duration of this apparent crossdesensitization in a second experiment by varying the delay between exposure to menthol and a block of three consecutive capsaicin stimuli. Cross-desensitization tended to decline as the interstimulus interval (ISI) increased to 5 min, and even when desensitization was maximal, it was significant only for the first of the three capsaicin stimuli. In the final experiment we investigated how menthol self- and cross-desensitization can influence the perception of menthol-capsaicin mixtures. During a series of five, 90-s stimulations, self- and cross-desensitization became evident at the beginning of the second exposure, but the effect on mixture intensity again diminished rapidly as stimulation continued. We infer from these results that method can transiently desensitize capsaicin-sensitive fibers, but that exposure to capsaicin rapidly overrides the effect. The implications these findings have for menthol's potential as a topical analgesic are discussed.

Thermal stimulation of taste.

The first electrophysiological recordings from animal and human taste nerves gave clear evidence of thermal sensitivity, and studies have shown that as many as half of the neurons in mammalian taste pathways respond to temperature. Because temperature has never been shown to induce sensations of taste, it has been assumed that thermal stimulation in the gustatory system is somehow nulled. Here we show that heating or cooling small areas of the tongue can in fact cause sensations of taste: warming the anterior edge of the tongue (chorda tympani nerve) from a cold temperature can evoke sweetness, whereas cooling can evoke sourness and/or saltiness. Thermal taste also occurs on the rear of the tongue (glossopharyngeal nerve), but the relationship between temperature and taste is different there than on the front of the tongue. These observations indicate the human gustatory system contains several different types of thermally sensitive neurons that normally contribute to the sensory code for taste.

Inhibition of bacterial peptide deformylase by biaryl acid analogs.

Peptide deformylase is an essential eubacterial metalloenzyme involved in the maturation of proteins by cleaving the N-formyl group from N-blocked methionine polypeptides. Biaryl acid analogs containing tetrazole, acyl sulfonamide, or carboxylate pharmacophores were found to be potent inhibitors of recombinant Escherichia coli peptide deformylase. Two of these compounds, a biphenyl tetrazole, compound 1, and a biphenyl acyl sulfonamide, compound 4, were competitive inhibitors with K(i) values of 1.2 and 6.0 microM, respectively. By analogy to the binding of related compounds to other metalloenzymes such as Bacteroides fragilis metallo-beta-lactamase CcrA and human carbonic anhydrase, a mechanism of inhibition is proposed for these peptide deformylase inhibitors where the acidic moieties form direct ionic interactions with the active site metal cation.

Sphincteric stent versus external sphincterotomy in spinal cord injured men: prospective randomized multicenter trial.

PURPOSE: In a prospective randomized multicenter trial we compared the treatment results of conventional external sphincterotomy with those of UroLume sphincteric stent prosthesis placement in men with spinal cord injury and external detrusor-sphincter dyssynergia. MATERIALS AND METHODS: We randomized 57 men with spinal cord injury in whom urodynamics verified external detrusor-sphincter dyssynergia into 2 groups to undergo either sphincter defeating procedure. We compared the primary urodynamic parameter of maximum detrusor pressure, and secondary urodynamic parameters of bladder capacity and post-void residual urine volume in men who underwent sphincterotomy or sphincteric stent placement. Parameters were measured preoperatively, and 3, 6, 12 and 24 months postoperatively. Patients completed questionnaires regarding voiding sensation and quality of life issues at each followup visit. RESULTS: Demographic data of the 26 patients treated with sphincterotomy and the 31 treated with sphincteric stent placement were statistically similar. Preoperatively mean maximum detrusor pressure plus or minus standard deviation in sphincterotomy and stent cases was 98.3 +/- 27.6 and 95.7 +/- 27.7 cm. water, respectively (p = 0.73). At 12 months mean maximum detrusor pressure decreased to 48.9 +/- 16.4 and 52.6 +/- 31.6 cm. water in the sphincterotomy and stent groups, respectively (p = 0). Preoperatively mean bladder capacity in sphincterotomy and stent cases was 245 +/- 158 and 251 +/- 145 ml., respectively (p = 0.87). Bladder capacity did not change significantly in either treatment group throughout followup. Preoperatively mean post-void residual urine volume in the sphincterotomy and stent groups was 212 +/- 163 and 168 +/- 114 ml., respectively (p = 0.33). Residual urine volume decreased in each group at some but not all followup evaluations. The duration of hospitalization was greater for sphincterotomy than stenting (p = 0.036). Six stents required explantation. CONCLUSIONS: The UroLume stent is as effective as conventional external sphincterotomy for treating external detrusor-sphincter dyssynergia. However, sphincteric stent placement is advantageous because it involves shorter hospitalization and is potentially reversible.

Temporal characteristics of capsaicin desensitization and stimulus-induced recovery in the oral cavity.

It was recently discovered that capsaicin desensitization of the tongue can be temporarily reversed during recurrent stimulation ("stimulus-induced recovery," SIR). The effects of concentration and the temporal pattern of stimulation on desensitization and SIR were studied in four experiments. In Experiment 1, three different concentrations of capsaicin (3.3, 33, or 330 microM) were delivered to the tongue in blocks of twelve stimuli at the rate of one per minute (treatment block) followed by a block of 20 exposures to 33 microM capsaicin (test block). The 33- and 330-microM treatments both caused significant desensitization, but by the end of the test block recovery was substantial only for the 33-microM condition. Experiment 2 provided a second test of the effect of concentration while also exploring whether intermittent stimulation was a requirement for SIR, i.e., stimuli were refreshed on the tongue every six minutes rather than every 60 s. SIR occurred but was pronounced only when the concentration of the test stimulus equaled or exceeded that of the treatment stimulus. In the third experiment we delivered capsaicin in a candy formulation to examine SIR under conditions in which stimulation was not interrupted at all. Although desensitization was evident during the first few minutes of succeeding exposures to the candy, peak irritation was unchanged throughout nine exposures over 3 days. In the final experiment, we returned to intermittent stimulation to examine the effect of lengthening ISI on SIR. The results showed that SIR remained virtually complete until ISI was lengthened to 60 s. We conclude that SIR is a robust phenomenon that is maximized by rapid exposure to capsaicin in a concentration at least as high as that of the desensitizing stimulus. The implications of these findings for hypotheses about the mechanism of SIR, its function in endogenous chemonociception, and for clinical use of capsaicin as a topical analgesic are discussed.

Structural requirements for human inducible nitric oxide synthase substrates and substrate analogue inhibitors.

Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) catalyzes the NADPH-dependent oxidation of one of the free guanidino nitrogens of L-Arg to form nitric oxide and L-citrulline. Analogues of L-Arg and the inhibitor, L-N6-(1-iminoethyl)lysine, were used to define structural elements required for the binding and catalysis of compounds. L-Arg analogues with sequentially shorter methylene spacing between the guanidino group and the amino acid portion of the molecule were not iNOS substrates but were reversible inhibitors. L-Arg analogues such as agmatine with a hydroxyl substitution at the 2-amino position were substrates. Desaminoarginine was not a substrate but a reversible inhibitor. Desaminoarginine, agmatine, and argininic acid bound to the enzyme to give type I difference spectra similar to that of L-Arg. The amidino compounds L-N6-(1-iminoethyl)lysine, L-N5-(1-iminoethyl)ornithine, and N5-(1-iminoethyl)cadaverdine, but not N6-(1-iminoethyl)-6-aminocaproic acid, were NADPH-dependent, irreversible inactivators of iNOS. For both the L-Arg and L-N6-(1-iminoethyl)lysine analogues, the 2-amino group appeared to play an important role in catalytic events leading to either substrate turnover or mechanism-based inactivation. Inactivation of iNOS by L-N6-(1-iminoethyl)lysine was NADPH- and dioxygen-dependent, but low incorporation of radiolabel with DL--4, 5-3H]-N6-(1-iminoethyl)lysine indicates that the mechanism of enzyme inactivation is not covalent modification of the protein.

Capsaicin desensitization and stimulus-induced recovery on facial compared to lingual skin.

It was recently demonstrated that capsaicin desensitization of the tongue can be temporarily reversed during bouts of recurrent or constant stimulation. The present study investigated whether this "stimulus-induced recovery" (SIR) also occurs on skin other than the oral mucosa. Twenty-two subjects received capsaicin treatments on the cheek and on the tongue tip at concentrations (330 and 33 microM) that produced approximately equal sensory irritation on the two sites. Desensitization and SIR occurred on both test sites, although the longer time course of irritation on the face changed the magnitude and form of SIR. There were large individual differences in the extent of desensitization and recovery, and the two phenomena were not correlated across sites, i.e., the capacity for SIR on the tongue was not a good predictor of an individual's capacity for SIR on the face. The results are discussed in terms of possible sources of regional and individual differences, and the implications they may have for the efficacy of topical analgesics that contain capsaicin.

"Warmth-insensitive fields": evidence of sparse and irregular innervation of human skin by the warmth sense.

Although more acute in some areas of the body than in others, temperature sensitivity is assumed to be present throughout the skin. Only when very small stimuli have been used (e.g., approximately 1 mm2) has sensitivity to warming or cooling appeared discontinuous. Here we report the discovery of patches of skin several square centimeters in area within which heating cannot be detected until skin temperature exceeds the thresholds of C heat-sensitive nociceptors (>41 degrees C). These warmth-insensitive fields (> or = 5 cm2), which appear to lack low-threshold warm fibers, were also found to have reduced responsiveness to non-painful heating and significantly higher heat pain thresholds compared to surrounding areas of skin. The existence of such sites corroborates reports that warm fibers are rare in human cutaneous nerves and confirms the classical theory that cutaneous innervation by the warmth sense is punctate and sparse. The insensitive areas also provide unique opportunities for assessing the contribution of the low-threshold warmth system to perception of heat and heat pain, and their existence in healthy young adults contraindicates use of warmth sensitivity in neurological assessments of C-fiber function.

Perceived irritation during ingestion of capsaicin or piperine: comparison of trigeminal and non-trigeminal areas.

The aim of this study was to investigate the perception of chemosensory irritation in the oropharyngeal region during the ingestion of irritants. In two experiments subjects sipped and swallowed small samples of an ascending concentration series of capsaicin or piperine and rated the intensity of sensations or irritation perceived at four locations: the anterior tongue, the posterior tongue, the roof of the mouth and the throat. Both experiments revealed that the responsiveness to irritation from capsaicin was significantly higher in the throat than at either the front or back of the tongue. There was no difference between irritation ratings for the throat and the roof of the mouth. Compared with capsaicin, the responsiveness to piperine was more uniform along the rostro-caudal axis; for example, irritation ratings for the throat were similar to those for the anterior tongue. These results support previous findings which indicated that the oral mucosae were not uniformly sensitive to chemical irritants, and suggest further that the throat, which is innervated by both the glossopharyngeal and vagus nerves, plays an important role in the perception of chemesthetic stimuli during ingestion.

Characterization of inducible nitric-oxide synthase by cytochrome P-450 substrates and inhibitors. Inhibition by chlorzoxazone.

Nitric-oxide synthases (NOS, EC 1.14.13.39) are heme-containing enzymes that catalyze the formation of nitric oxide from L-Arg. General cytochrome P-450 inhibitors and cytochrome P-450 isoform-selective substrates and inhibitors were used to characterize the activity of recombinant human inducible NOS (iNOS). Classical cytochrome P-450 ligands such as the mechanism-based inactivator 1-aminobenzotriazole did not inhibit iNOS. Of a panel of 30 human cytochrome P-450 isoform-selective substrates and inhibitors, only chlorzoxazone, a cytochrome P-450 2E1 (CYP2E1) substrate, showed any significant inhibition of iNOS activity. Chlorzoxazone was not a substrate for iNOS but was a potent competitive inhibitor with respect to L-Arg with Ki = 3.3+/-0.7 microM. The binding of chlorzoxazone to iNOS and human and rat liver microsomal cytochrome P-450 induced a high spin, type I spectra, which was reversed by imidazole. Although the binding of chlorzoxazone to iNOS and its inhibition of iNOS activity suggest some similarity between iNOS and CYP2E1 activity, other CYP2E1 substrates and inhibitors including zoxazolamine were not inhibitors of iNOS. Overall, iNOS activity is distinctly different from the major cytochrome P-450 enzymes in human liver microsomes.

Rapid recovery from capsaicin desensitization during recurrent stimulation.

Topical desensitization of the tongue was assessed during multiple bouts of exposure to capsaicin. In the first experiment subjects rated perceived irritation as 30 capsaicin stimuli (33 microM) were applied to the tongue tip in three blocks of 10, with 15 min breaks between blocks. Significant desensitization was measured at the beginning of the second and third blocks within each session. However, as stimulation continued within those blocks sensations of irritation grew toward undesensitized levels ('stimulus-induced recovery' (SIR)). Desensitization did not extend across days. The second experiment employed a 10-fold higher concentration of capsaicin (330 microM) to determine if SIR was limited to low levels of desensitization. SIR occurred as before within sessions, and the higher concentration produced desensitization across days that also exhibited recovery during the first block of stimuli on days 2 and 3. The third experiment included piperine, zingerone and citric acid as stimuli to determine if SIR was specific to capsaicin. Piperine produced SIR under conditions of both self- and cross-desensitization with capsaicin, whereas recovery failed to materialize with zingerone. Citric acid was not significantly cross-desensitized by capsaicin, so recovery could not be measured. Overall the results demonstrate that desensitization of the tongue produced by either capsaicin or piperine can be temporarily reversed if stimulation with either chemical is resumed for only a few minutes. The implications these findings may have for hypotheses about the mechanisms of capsaicin desensitization and sensitization as well as for clinical applications of capsaicin as a topical analgesic are discussed.

The natural product noformycin is an inhibitor of inducible-nitric oxide synthase.

Inducible-Nitric oxide synthase (iNOS, EC 1.14.13.39) catalyzes the formation of nitric oxide (NO) and L-citrulline from L-Arg. NADPH and dioxygen. The natural product, (-)-noformycin was found to be a potent, competitive inhibitor of recombinant human iNOS with respect to L-Arg with a Ki = 1.3 +/- 0.3 microM. The reversible binding of noformycin caused a high spin type I spectral perturbation of the iNOS heme group with a Kd = 1.5 +/- 0.2 microM. These results demonstrate that natural products may be a useful source for inhibitors of NO-biosynthesis.