Activation of purinergic P2X receptors associated with the parasympathetic nerves that supply the human bladder smooth muscle (detrusor) is implicated in control of detrusor contractility. The relative abundance of all seven subtypes colocalized with synaptic vesicles on parasympathetic nerves was examined in specimens from normal adult bladder, infants, and in adults with overactive detrusor contractility and a diagnosis of idiopathic detrusor instability (IDI) to determine whether receptor distribution varied with age or in patients with incontinence. Alteration in control of detrusor innervation was examined with P2X subtype-specific antibodies and an antibody against synaptic vesicles, using immunofluorescence and confocal microscopy. Detrusor samples were taken from: controls, at cystectomy for cancer or cystoscopic biopsy for hematuria (n = 22; age 33-88), child bladder, at surgical correction of vesico-ureteric reflux (n = 21; age 4 months to 2 years), and adults with detrusor instability at cystoscopy-cystodistension (n = 18; age 30-81). Adult specimens contained muscle with large varicosities (1.2 microm) along parasympathetic nerves with colocalized patches of all P2X(1-7) subtypes. Infant bladder revealed little evidence of P2X at age <9 months but approached adult levels at 2 years. Detrusor from IDI patients revealed selective absence of P2X(3) and P2X(5) beneath all the varicosities. This specific lack of P2X(3) and P2X(5) may impair control of detrusor contractility and contribute to the pathophysiology of urge incontinence.

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The structure of the biologically active N-terminal domain of human parathyroid-hormonerelated protein (residues 1 -34) containing an Ala substituted for a His in position 26 was studied by two-dimensional proton NMR spectroscopy. Unambiguous NMR assignments of all backbone and side-chain hydrogens were made with the aid of totally correlated spectroscopy experiments, which provided through-bond 'H-'H connectivities, and NOE spectroscopy, which provided through-space and sequential backbone connectivities. The NMR data were utilized in distance-geometry algorithms to generate a family of structures. The major structural features include two segments of a-helix extending from Glu4 to Lysl3 and from Leu27 to Thr33, with two turns from Gln16 to Argl9 and Phe22 to His25. A salt-bridge appears likely between Arg20 and Glu30 which may be critical for holding the receptor-binding domain together.Humoral hypercalcemia of malignancy is a common complication of certain cancers caused by the secretion of the human parathyroid-hormone-like protein. Peptides secreted by human tumors associated with humoral hypercalcemia of malignancy [l, 21 recently have been characterized [3-51. They exhibit a high affinity for both renal and skeletal parathyroid-hormone (PTH) receptors [6 -81. Sequence studies have shown that there is significant similarity between these hypercalcemia associated peptides and human PTH, particularly in the N-terminal region where 8 of the first 13 residues are identical [I]. For this reason these peptides have been termed parathyroid-hormone-related protein, PTHrP. There is no sequence similarity between PTHrP and PTH in the Cterminal region. PTHrP mimicks the major biological actions of PTH [6, 8 -111. PTH(1-34) and PTHrP(1-34) exhibit the same activity as shown by the native PTH(1-84) and PTHrP(1-141) as measured in a range of assays [6, 9, 121. Detailed comparisons between the binding of these native peptide hormones and various analogues to PTH receptors have provided a much deeper insight into both the location of the receptor-binding site and the structural basis of agonist activity. A preliminary NMR study of PTHrP indicated a surprisingly compact structure [13] and a recent CD study has indicated a helix/helix interaction, especially in the presence of an amphiphilic solvent [14]. We have synthesized several analogues of PTHrP(1-34) containing single amino acid substitutions as a means of determining the role of various resi- trifluoroethanol to provide a more refined model of the biologically active fragment in solution. This work has confirmed that the structure in the presence of trifluoroethanol is largely stabilized, exhibiting an N-terminal helix and a hydrophobic core in the C-terminal domain. A C-terminal sequence of CIhelix is observed from Leu27 to Thr33 together with two bends. Some structural basis is provided for understanding the large differences in agonist activity exhibited by smaller peptide fragments in a range of assays. MATERIALS AND METHODS Peptide synthesesPTHrP(1-34) analogue...

Purinergic P2X receptors associated with the parasympathetic nerves supplying human bladder smooth muscle (detrusor) are implicated in control of detrusor contractility. The relative abundance of all seven subtypes colocalised with synaptic vesicles on parasympathetic nerves was examined in specimens from normal adult bladder and in adults with the urodynamics findings of sensory urgency (SU) to determine how receptor distribution varied in patients with a small bladder capacity. Alteration in control of detrusor innervation was examined with P2X subtype-specific antibodies and an antibody (SV2) against synaptic vesicles, using immunofluorescence and confocal microscopy. Detrusor samples were taken from: controls, at cystectomy for cancer or cystoscopic biopsy for haematuria (n=22, age 33-88 years) and adults with sensory urgency at cystoscopy/cystodistension (n=11, age 37-70 years). Normal adult specimens contained detrusor muscle innervated by parasympathetic nerves possessing large varicosities (1.2 microm) distributed along their length. These mostly all showed colocalised patches of presynaptic P2X(1,2,3,5) subtypes while presynaptic subtypes P2X(4,6,7) were present in only 6-18% of varicosities. Detrusor nerve varicosities from SU patients revealed general loss of all presynaptic P2X subtypes with the proportion containing receptors reducing to only 0.5-5% depending on P2X subtype. The same loss was recorded from the sensory nerves in the surrounding lamina propria. This specific loss of P2X receptors may impair control of detrusor distension and contribute to the pathophysiology of sensory urgency.

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