The PCAST report: impact and implications for the pharmaceutical industry.

The President's Council of Advisors on Science and Technology (PCAST) report sets out an ambitious goal: to double the output of innovative, new medicines with increased efficacy and safety within the next 10-15 years. If attainable, this could change the face of medicine and bring great benefit to society. Clear leadership, commitment to action, and unprecedented collaboration will be essential if the goal of the report is to be realized.

Two-year results of once-weekly administration of alendronate 70 mg for the treatment of postmenopausal osteoporosis.

The aim of this study was to provide confirmation that once-weekly dosing with 70 mg of alendronate (seven times the daily oral dose) and twice-weekly dosing with 35 mg is equivalent to the 10-mg once-daily regimen and to gain more extensive safety experience with this new dosing regimen. Twelve hundred fifty-eight postmenopausal women (aged 42-95 years) with osteoporosis (bone mineral density [BMD] of either lumbar spine or femoral neck at least 2.5 SDs below peak young adult mean or prior vertebral or hip fracture) were assigned to receive oral once-weekly alendronate, 70 mg (n = 519); twice-weekly alendronate, 35 mg (n = 369); or daily alendronate 10 mg (n = 370) for a total of 2 years of double-blind experience. Mean BMD increases from baseline (95% CI) at 24 months in the once-weekly, twice-weekly, and daily treatment groups, respectively, were 6.8% (6.4, 7.3), 7.0% (6.6,7.5), and 7.4% (6.9,7.8) at the lumbar spine and 4.1% (3.8,4.5), 4.3% (3.9,4.7), and 4.3% (3.9,4.7) at the total hip. These increases in BMD as well as the BMD increases at the femoral neck, trochanter, and total body and the reductions of biochemical markers of bone resorption (urinary cross-linked N-telopeptides of type I collagen [NTx]) and bone formation (serum bone-specific alkaline phosphatase [BSAP]) were similar for the three dosing regimens. All treatment regimens were well tolerated with a similar incidence of upper gastrointestinal (GI) adverse experiences. The incidence rates of clinical fractures, captured as adverse experiences, were similar among the groups. The 2-year results confirm the conclusion reached after 1 year that once-weekly alendronate is therapeutically equivalent to daily dosing, providing patients with a more convenient dosing option that may potentially enhance adherence to therapy.

Therapeutic equivalence of alendronate 70 mg once-weekly and alendronate 10 mg daily in the treatment of osteoporosis. Alendronate Once-Weekly Study Group.

Dosing convenience is a key element in the effective management of any chronic disease, and is particularly important in the long-term management of osteoporosis. Less frequent dosing with any medication may enhance compliance, thereby maximizing the effectiveness of therapy. Animal data support the rationale that once-weekly dosing with alendronate 70 mg (7 times the daily oral treatment dose) could provide similar efficacy to daily dosing with alendronate 10 mg due to its long duration of effect in bone. In addition, dog studies suggest that the potential for esophageal irritation, observed with daily oral bisphosphonates, may be substantially reduced with once-weekly dosing. This dosing regimen would provide patients with increased convenience and would be likely to enhance patient compliance. We compared the efficacy and safety of treatment with oral once-weekly alendronate 70 mg (N=519), twice-weekly alendronate 35 mg (N=369), and daily alendronate 10 mg (N=370) in a one-year, double-blind, multicenter study of postmenopausal women (ages 42 to 95) with osteoporosis (bone mineral density [BMD] of either lumbar spine or femoral neck at least 2.5 SDs below peak premenopausal mean, or prior vertebral or hip fracture). The primary efficacy endpoint was the comparability of increases in lumbar spine BMD, using strict pre-defined equivalence criteria. Secondary endpoints included changes in BMD at the hip and total body and rate of bone turnover, as assessed by biochemical markers. Both of the new regimens fully satisfied the equivalence criteria relative to daily therapy. Mean increases in lumbar spine BMD at 12 months were: 5.1% (95% CI 4.8, 5.4) in the 70 mg once-weekly group, 5.2% (4.9, 5.6) in the 35 mg twice-weekly group, and 5.4% (5.0, 5.8) in the 10 mg daily treatment group. Increases in BMD at the total hip, femoral neck, trochanter, and total body were similar for the three dosing regimens. All three treatment groups similarly reduced biochemical markers of bone resorption (urinary N-telopeptides of type I collagen) and bone formation (serum bone-specific alkaline phosphatase) into the middle of the premenopausal reference range. All treatment regimens were well tolerated with a similar incidence of upper GI adverse experiences. There were fewer serious upper GI adverse experiences and a trend toward a lower incidence of esophageal events in the once-weekly dosing group compared to the daily dosing group. These data are consistent with preclinical animal models, and suggest that once-weekly dosing has the potential for improved upper GI tolerability. Clinical fractures, captured as adverse experiences, were similar among the groups. We conclude that the alendronate 70 mg once-weekly dosing regimen will provide patients with a more convenient, therapeutically equivalent alternative to daily dosing, and may enhance compliance and long-term persistence with therapy.

Immunohistochemical detection of parathyroid hormone-related protein in a cutaneous squamous cell carcinoma causing humoral hypercalcemia of malignancy.

Humoral hypercalcemia of malignancy is a cancer-related hypercalcemia caused by production of humoral factors by malignant cells in patients without bone metastases. Squamous cell carcinomas are the tumors most frequently associated with humoral hypercalcemia of malignancy, and parathyroid hormone-related protein is the main humoral factor implicated. In spite of the fact that normal keratinocytes produce parathyroid hormone-related protein, it is highly unusual for patients with squamous cell carcinomas of the skin to present with humoral hypercalcemia of malignancy. We present a well-documented case of cutaneous squamous cell carcinoma complicated by hypercalcemia in a patient with high levels of plasma parathyroid hormone-related protein and immunohistochemical evidence of high parathyroid hormone-related protein production by the tumoral cells.

Expression and role of parathyroid hormone-related protein in human renal proximal tubule cells during recovery from ATP depletion.

Parathyroid hormone (PTH)-related protein (PTHrP) is widely expressed in normal fetal and adult tissues and regulates growth and differentiation in a number of organ systems. Although various renal cell types produce PTHrP, and PTHrP expression in rat proximal renal tubules is upregulated in response to ischemic injury in vivo, the role of PTHrP in the kidney is unknown. To study the effects of injury on PTHrP expression and its consequences in more detail, the immortalized human proximal tubule cell line HK-2 was used in an in vitro model of ATP depletion to mimic in vivo renal ischemic injury. These cells secrete PTHrP into conditioned medium and express the type I PTH/PTHrP receptor. Treatment of confluent HK-2 cells for 2 h with substrate-free, glucose-free medium containing the mitochondrial inhibitor antimycin A (1 microM) resulted in 75% depletion of cellular ATP. After an additional 2 h in glucose-containing medium, cellular ATP levels recovered to approximately 75% of baseline levels. PTHrP mRNA levels, as measured in RNase protection assays, peaked at 2 h into the recovery period (at four times baseline expression). The increase in PTHrP mRNA expression was correlated with an increase in PTHrP protein content in HK-2 cells at 2 to 6 h into the recovery period. Heat shock protein-70 mRNA expression was not detectable under baseline conditions but likewise peaked at 2 h into the recovery period. Treatment of HK-2 cells during the recovery period after injury with an anti-PTHrP(1-36) antibody (at a dilution of 1:250) resulted in significant reductions in cell number and uptake of [3H]thymidine, compared with nonimmune serum at the same titer. Similar results were observed in uninjured HK-2 cells. It is concluded that this in vitro model of ATP depletion in a human proximal tubule cell line reproduces the pattern of gene expression previously observed in vivo in rat kidney after ischemic injury and that PTHrP plays a mitogenic role in the proliferative response after energy depletion.

Stromal cells are critical targets in the regulation of mammary ductal morphogenesis by parathyroid hormone-related protein.

Parathyroid hormone-related protein (PTHrP) was originally identified as the tumor product responsible for humoral hypercalcemia of malignancy. It is now known that PTHrP is produced by many normal tissues in which it appears to play a role as a developmental regulatory molecule. PTHrP is a normal product of mammary epithelial cells, and recent experiments in our laboratory have demonstrated that overexpression or underexpression of PTHrP in the murine mammary gland leads to severe disruptions in its development. The nature of these phenotypes suggests that PTHrP acts to modulate branching growth during mammary development by regulating mammary stromal cell function. We now demonstrate that throughout mammary development, during periods of active ductal-branching morphogenesis, PTHrP is produced by epithelial cells, whereas the PTH/PTHrP receptor is expressed on stromal cells. In addition, we show that mammary stromal cells in culture contain specific binding sites for amino terminal PTHrP and respond with an increase in intracellular cAMP. Finally, we demonstrate that the mammary mesenchyme must express the PTH/PTHrP receptor in order to support mammary epithelial cell morphogenesis. These results demonstrate that PTHrP and the PTH/PTHrP receptor represent an epithelial/mesenchymal signaling circuit that is necessary for mammary morphogenesis and that stromal cells are a critical target for PTHrP's action in the mammary gland.

A midregion parathyroid hormone-related peptide mobilizes cytosolic calcium and stimulates formation of inositol trisphosphate in a squamous carcinoma cell line.

A midregion fragment of PTH-related protein (PTHrP), which is intensively conserved across species, has been identified as a secretory product of several different cell types, including keratinocytes and squamous carcinomas. As recent data suggest that a midregion PTHrP fragment may be biologically active, we hypothesized that midregion PTHrPs interact with unique cell surface receptors that mediate autocrine or paracrine action. Dose-dependent transient elevations in intracellular calcium ([Ca2-]i) were observed in fura-2-loaded SqCC/Y1 squamous carcinoma cells exposed to human (h) PTHrP-(67-86)NH2, [Tyr36]hPTHrP-(1-36)NH2, and hPTHrP-(1-141) at concentrations ranging from 1 pM to 1 microM. The effects of maximal stimulatory concentrations of [Tyr36]PTHrP-(1-36)NH2 and PTHrP-(67-86)NH2 on [Ca2+]i were additive. The inhibitory PTH analog, [D-Trp12,Tyr34]bovine PTH-(7-34)NH2, attenuated the [Ca2+]i response to [Tyr36]hPTHrP-(1-36)NH2, but not that to PTHrP-(67-86)NH2. These data suggest that PTHrP-(67-86)NH2 activates a different receptor pathway in SqCC/Y1 cells from the one activated by [Tyr36]hPTHrP-(1-36)NH2. Radiolabeled PTHrP-(67-86)NH2 did not bind to SqCC/Y1 cells, and PTHrP-(67-86)NH2 did not compete for binding of 125I-labeled [Tyr36]PTHrP-(1-36)NH2 to PTH/PTHrP receptors on SaOS-2 osteosarcoma cells. Activation of the phospholipase C pathway by PTHrP-(67-86)NH2 was confirmed by exposing SqCC/Y1 cells to peptide for 1 min and measuring the accumulation of inositol trisphosphates. PTHrP-(67-86)NH2 treatment (100 nM) resulted in maximal stimulation of inositol trisphosphates of 3.1 +/- 0.1-fold over the control value, with an EC50 of 1.5 +/- 1.2 nm. In contrast, PTHrP-(67-86)NH2 (0.1 nM to 1 microM) did not stimulate adenylyl cyclase in SqCC/Y1 cells despite vigorous stimulation of cAMP formation by isoproterenol (1 microM) to 66-fold over the basal value. To determine whether messenger RNA (mRNA) prepared from SqCC/Y1 cells would direct the translation of a receptor protein that mediated a [Ca2+]i response to PTHrP-(67-86)NH2, we performed expression studies in Xenopus oocytes. Fluo-3 fluorescence in Xenopus oocytes expressing SqCC/Y1 mRNA was visualized by confocal video microscopy after exposure to 1 microM PTHrP-(67-86)NH2. Clear increases in [Ca2+]i were detected in mRNA-injected, but not in sham-injected, oocytes. Finally, we examined the effect of PTHrP-(67-86)NH2 treatment on fibronectin secretion from SqCC/YN1 cells. A significant 3.5-fold increase in fibronectin secretion into conditioned medium was observed when SqCC/Y1 cells were exposed to 100 nM PTHrP-(67-86)NH2, and this effect was dose dependent, with an EC50 of 0.1 nM. We conclude that PTHrP-(67-86)NH2 activates phospholipase C-dependent pathways in SqCC/Y1 cells through a receptor distinct from that activated by PTHrP-(1-36) in the same cells. As a midregion secretory fragment of PTHrP has been partially purified from several different cell types, this receptor may have broad biological significance.

Clonal variation of p53 expression and proliferative phenotype in A253 squamous carcinoma cells.

Loss of normal p53 tumor-suppressor gene function is characteristic of the majority of squamous carcinomas. During the course of gene transfer studies in the human squamous carcinoma cell line, A253, which does not express p53 mRNA or protein, we incidentally observed increased levels of p53 expression in up to 20% of clonal cell lines derived from parental A253 cells. p 53-expressing A253 cells (A253-p53) were also isolated by dilutional cloning. Nuclear p53 protein was identified by immunohistochemistry in A253-p53 cells in a wild-type pattern, and p53 mRNA (2.5 kb) was demonstrated by northern blot. Mutational analysis of the p53 gene in A253-p53 cells revealed no evidence for mutations in exons 5-9. A253-p53 cells could be distinguished from native A253 cells by prolonged doubling times (2-5 fold) and by a marked reduction of [3H]-thymidine uptake. Whereas A253 cells were unresponsive to the growth-inhibitory effects of TGF-beta, EGF-stimulated A253-p53 cells responded to TGF-beta with markedly reduced DNA synthetic rates. A253-p53 cells cocultured with A253 demonstrated enhanced cell growth and DNA synthesis rates compared to control A253-p53 cells. Finally, A253-p53 cells show reduced expression of c-fos, fibronectin, thrombospondin and parathyroid hormone-related protein (PTHrP) mRNAs. PTHrP measured by RIA in conditioned medium was approximately 300 pM for A253 but undetectable for A253-p53. We conclude that the A253 cell line contains a subpopulation of cells which express high levels of "wild-type-like" p53 protein. This results in dramatic changes in gene expression and a slower-growing phenotype in vitro.

Defining the roles of parathyroid hormone-related protein in normal physiology.

Parathyroid hormone-related protein (PTHrP) was discovered as a result of a search for the circulating factor secreted by cancers which causes the common paraneoplastic syndrome humoral hypercalcemia of malignancy. Since the identification of the peptide in 1982 and the cloning of the cDNA in 1987, it has become clear that PTHrP is a prohormone that is posttranslationally cleaved by prohormone convertases to yield a complex family of peptides, each of which is believed to have its own receptor. It is also clear that the PTHrP gene is expressed not only in cancers but also in the vast majority of normal tissues during adult and/or fetal life. In contrast to the situation in humoral hypercalcemia of malignancy in which PTHrP plays the role of a classical "endocrine" hormone, under normal circumstances PTHrP plays predominantly paracrine and/or autocrine roles. These apparent physiological functions are also complex and appear to include 1) regulation of smooth muscle (vascular, intestinal, uterine, bladder) tone, 2) regulation of transepithelial (renal, placental, oviduct, mammary gland) calcium transport, and 3) regulation of tissue and organ development, differentiation, and proliferation. In this review, the discovery of PTHrP, the structure of its gene and its cDNAs, and the posttranslational processing of the initial translation products are briefly reviewed. Attention is then focused on a detailed organ system-oriented review of the normal physiological functions of PTHrP.

Further evidence for a novel receptor for amino-terminal parathyroid hormone-related protein on keratinocytes and squamous carcinoma cell lines.

PTH and PTH-related peptides (PTHrPs) interact with a common PTH/PTHrP receptor (type I), which is expressed in many tissues, including bone and kidney. Amino-terminal PTH and PTHrPs also recognize receptors in several nonclassical PTH target tissues, and in some of these, the signaling mechanisms differ qualitatively from those of the classical type I receptor. In normal keratinocytes and squamous carcinoma cell lines, PTH and PTHrP stimulate a rise in intracellular calcium, but not cAMP, suggesting the existence of an alternate, type II PTH/PTHrP receptor. SqCC/Y1 squamous carcinoma cells stably expressing the type I receptor displayed sensitive intracellular cAMP responses to PTHrP and PTH, indicating that these cells express functional GS proteins and that the type I receptor is capable of signaling through adenylyl cyclase in this cell line. Therefore, the endogenous type II receptor in SqCC/Y1 cells differs from the cloned type I receptor. We next examined whether messenger RNA (mRNA) from keratinocytes and squamous cell lines could hybridize to a human type I PTH/PTHrP receptor complementary DNA [1.9 kilobases (kb)]. No type I receptor mRNA (2.3 kb) was detected in polyadenylated RNA from any of the squamous cell lines. However, squamous cell lines did express several mRNA transcripts that hybridized with the type I receptor probe, yet were smaller (1 and 1.5 kb) or larger (3.5-5 kb) than the cloned receptor mRNA. The predominant mRNA in two squamous carcinoma cell lines and normal keratinocytes was a 1-kb transcript. Northern analysis with five different region-specific probes that span the entire coding region of the human type I receptor was used to map homologous regions within each of the transcripts. Several of the transcripts identified in squamous lines are also present in polyadenylated RNA from SaOS-2 human bone cells, but a unique 1-kb transcript hybridizing to probe 2 (nucleotides 490-870) was observed only in squamous cells. The smaller 1- and 1.5-kb transcripts did not hybridize to probes corresponding to the extreme 5'- and 3'-coding regions of the type I receptor complementary DNA. Ribonuclease protection analysis employing riboprobes that correspond to the five region-specific DNA probes revealed strong RNA signals of the expected size in SaOS-2 cells, but no hybridization with squamous cell RNA. Several smaller, but minor, bands that were unique to squamous cells were observed with riboprobe 2 only, suggesting partial homology of this region with the type I receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of desensitization of the cloned vasopressin V1a receptor expressed in Xenopus oocytes.

The vasopressin V1a receptor exerts its effects by G protein-mediated increases in cytosolic Ca2+ (Cai2+) and activation of protein kinase C. The V1a receptor also undergoes autologous desensitization. To clarify the mechanism of this desensitization, we expressed the cloned receptor in Xenopus oocytes, and vasopressin-induced Cai2+ waves were examined as an index of V1a activation using confocal microscopy. Pretreatment of oocytes with a minimal concentration of vasopressin inhibited further generation of Cai2+ waves upon maximal stimulation. Such pretreatment did not abolish Cai2+ waves induced by subsequent microinjection of inositol trisphosphate, suggesting that this phenomenon represents receptor desensitization rather than depletion of inositol trisphosphate-sensitive Cai2+ stores. Pretreatment with phorbol dibutyrate, ionomycin, or 8-bromoadenosine 3',5'-cyclic monophosphate had no effect on vasopressin-induced Cai2+ waves. Oocytes recovered from desensitization within 1 h, but the microtubule inhibitor methyl-5-[2-thienylcarbonyl]-1H-benzimiidazol-2-yl)-carbamate (nocodazole) inhibited this recovery. Receptor binding sites were reduced by over 50% within 10 min of exposure to vasopressin, with no associated change in the Kd for the V1a receptor. These findings indicate that 1) expression of the cloned V1a receptor in Xenopus oocytes, coupled with subcellular Cai2+ imaging, provides a useful system to examine mechanisms of V1a desensitization, 2) the V1a receptor undergoes autologous desensitization in this experimental system, and 3) protein kinase C, Cai2+, and adenosine 3',5'-cyclic monophosphate do not appear responsible for this desensitization, but 4) microtubule-dependent recycling of the receptor is preserved in this system and may be important for receptor desensitization.

Parathyroid hormone-related protein as a prohormone: posttranslational processing and receptor interactions.

Since the elucidation of the structures of the three human PRHrP isoforms in 1987, information has rapidly accured which indicates that the role of PTHrP in normal physiology will prove to be crucial as well as exceedingly complex. The importance of the role of PTHrP in normal physiology is underscored by its broad tissue expression, by its intense evolutionary conservation, by its extremely early expression after fertilization of the ovum, and by the lethal consequences of PTHrP gene disruption. The complexity of the role of PTHrP in normal physiology increases almost monthly. This complexity is reflected in the broad tissue distribution of the peptide, its complex transcriptional regulation and mRNA instability motifs, and its multiple transcripts and isoforms. It is now clear that additional complexity exists at the level of posttranslational processing. Expression of the PTHrP gene leads to the tissue-specific processing and secretion of an increasingly complex family of derivative peptides, each with its own repertoire of cognate receptors, signal transduction pathways, and physiological consequences. Further elucidation of the posttranslational processing pathways and mechanisms can be anticipated in the coming years, coupled with a corresponding elucidation of multiple PTHrP receptors, their specific signal transduction pathways, and their unique physiological roles. The role of PTHrP in causing HHM is now clearly established. Work in the coming decade will focus on the normal physiological roles played by PTHrP.

Accumulation of carboxy-terminal fragments of parathyroid hormone-related protein in renal failure.

We have recently demonstrated elevations of separate amino- and carboxy-terminal parathyroid hormone-related protein (PTHrP) fragments in patients with humoral hypercalcemia of malignancy (HHM) using both a two-site immunoradiometric assay (IRMA) with amino-terminal specificity for PTHrP, and with a carboxy-terminal radioimmunoassay (RIA) for PTHrP(109-138). PTHrP(109-138) immunoactivity from plasma of patients with HHM could not be extracted using an amino-terminal PTHrP immunoaffinity column, indicating that the carboxy-terminal region circulates as a discrete peptide. Carboxy-terminal immunoreactive (i) PTHrP levels were also elevated in normocalcemic patients with chronic renal failure (without cancer), whereas amino-terminal iPTHrP levels were normal in patients with renal failure. In order to further define the renal handling of carboxy-terminal PTHrP peptides, we have evaluated circulating iPTHrP(109-138) concentrations in patients with a wide range of renal function. We studied 25 patients with abnormal renal function of diverse etiologies whose creatinine clearances ranged from 66 ml/min to less than 5 ml/min. All patients had undetectable or low (< or = 2 pmol/liter) concentrations of iPTHrP(1-74). iPTHrP(109-138) concentrations were undetectable in patients with creatinine clearances > or = 20 ml/min, but became elevated in patients with creatinine clearances < 20 ml/min. The log of iPTHrP(109-138) correlated negatively with the log of creatinine clearance (r = 0.88, P = 0.0001). Mean iPTHrP(109-138) levels were slightly higher for patients on hemodialysis (32.7 +/- 3.1 pM) than for those on chronic ambulatory peritoneal dialysis (22.1 +/- 3.4 pM; P < 0.05), suggesting that some carboxy-terminal PTHrP fragments may be cleared to a greater extent by the peritoneal membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino-terminal parathyroid hormone-related protein: specific binding and cytosolic calcium responses in rat insulinoma cells.

PTH-related protein (PTHrP), originally identified through its causative role in human humoral hypercalcemia of malignancy, is now known to be a normal gene product expressed in a wide variety of neuroendocrine, epithelial, and mesoderm-derived tissues. PTHrP gene expression has recently been demonstrated in fetal and adult, benign and malignant, as well as human and rodent pancreatic islets. As in other tissues, the role of PTHrP expression in the normal islet is only beginning to be explored. In the current report, PTHrP expression in the normal rat pancreatic islet was confirmed using an affinity-purified antiserum directed against the N-terminal, biologically active region of the molecule. The effects of PTHrP on the islet were then explored using rat insulinoma (RIN m5F) cells. Synthetic PTHrP-(1-36) bound specifically, but with low affinity (Kd, approximately 10(-7) M) to RIN cell membranes. PTHrP-(1-36) failed to stimulate cAMP production in RIN cells, although RIN cells displayed a normal adenylate cyclase response to glucagon-like peptide-1-(7-36). In contrast, PTHrP-(1-36) induced a rapid dose-dependent rise in intracellular calcium in RIN cells in doses as low as 10(-12)-10(-10) M. These findings 1) confirm that PTHrP is expressed by islet cells, 2) demonstrate that the effects of PTHrP on the pancreatic islet are mediated, as in keratinocytes and lymphocytes, by a receptor related to but distinct from the PTH receptor, and 3) suggest that PTHrP functions in the islet as an autocrine or paracrine factor. Further studies are required to determine the physiological consequences of PTHrP expression by the pancreatic islet.

A pharmacological comparison of parathyroid hormone receptors in human bone and kidney.

While abundant information is available characterizing PTH receptor properties in other species, data on human PTH receptors is very limited. We have been interested in the possibility that tissue-specific differences among human PTH receptors (i.e. bone vs. kidney) might exist. We have, therefore, compared pharmacological profiles for a wide array of PTH and PTH-related peptide (PTHrP) analogs in human osteoblast-like cells (SaOS-2) and human renal cortical membranes (RCM) using radioiodinated (Tyr36)hPTHrP(1-36)NH2 as a probe for PTH receptor function. The rank order of receptor affinity for 10 PTH/PTHrP receptor agonists tested was very similar in the bone and kidney assay systems. Binding affinity for these peptides was greater in human (h)RCMs and SaOS-2 membranes than in SaOS-2 intact cells. The relative binding affinities for (Tyr36)hPTHrP(1-36)amide, hPTH(1-34), bovine (b)PTH(1-34), and rat PTH(1-34) were similar in human RCMs, SaOS-2 membranes, and SaOS-2 cells. bPTH(1-84) and hPTHrP(1-74) both manifested lower receptor affinity than the amino-terminal analogs. Seven PTH/PTHrP receptor antagonists were also studied in this homologous human assay system. The binding affinity for hPTHrP(7-34)NH2 was 2- to 3-fold greater than that for (Tyr34)bPTH(7-34)NH2 in RCM and SaOS-2 membranes. However, in SaOS-2 cells, a striking reversal in the relative binding affinities was observed; the PTHrP(7-34) analog was nearly 3-fold less potent than (Tyr34)bPTH(7-34)NH2, underscoring a difference between intact and broken cell preparations. Two hybrid PTH-PTHrP receptor antagonists demonstrated similar relative affinity to each other in the human bone and kidney assay systems. Affinity cross-linking of receptors in human renal and skeletal tissues demonstrated an indistinguishable dominant 85-kilodalton receptor protein. We conclude that the binding and bioactivity profiles of a broad array of PTH and PTHrP peptides are very similar or identical in human renal and skeletal tissues. Differences relating to intact vs. broken cell preparations accounted for some variation in potency. These studies emphasize the importance of employing homologous assay systems to study PTH receptor function and the existence of interspecies differences among PTH receptors. The results support the possibility that PTH receptors in human bone and human kidney are very similar if not identical.

Analysis of PTHRP binding and signal transduction mechanisms in benign and malignant squamous cells.

We have explored a potential autocrine role for parathyroid hormone-related protein (PTHRP) in malignant squamous carcinoma cells (SqCC) and their nonmalignant counterpart, human epidermal keratinocytes (HK). Specific binding of Tyr36 human PTHRP-(1-36)NH2 (125I-[Tyr36]hPTHRP-(1-36)NH2) was identified in 75% of unselected SqCC lines. In contrast, no binding was detected on the mouse keratinocyte line BALB-MK or on five different HK lines. Although each SqCC and keratinocyte line secreted immunoreactive PTHRP into its medium, there was no correlation between PTHRP concentration and number of binding sites. Inhibition of binding by [Tyr36]hPTHRP-(1-36)NH2 yielded half-maximal inhibitory concentration values of approximately 100 nM in all SqCC lines. Affinity cross-linking of SqCC cells revealed 98- and 70-kDa binding proteins with similar affinity (approximately 100 nM). Exposure of fura-2-loaded SqCC cells to PTHRP and PTH resulted in equivalent, dose-dependent transient increases in intracellular calcium [half-maximal effective concentration (EC50) = 0.08 nM]. PTHRP also increased intracellular calcium in HK (EC50 = 0.05 nM). No adenosine 3',5'-cyclic monophosphate (cAMP) response to PTHRP or PTH was elicited in either SqCC or HK, despite brisk isoproterenol responses in both. We conclude that high-capacity low-affinity binding sites for PTHRP are detectable in the majority of SqCC lines but not in HK. These low-affinity binding sites are unlikely to represent receptors. The sensitive intracellular calcium response suggests the additional presence of high-affinity receptors on SqCC as well as on HK. However, the failure of PTHRP or PTH to stimulate cAMP production in otherwise cyclase-competent cells suggests that these are not classical PTH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythromycin ototoxicity: prospective assessment with serum concentrations and audiograms in a study of patients with pneumonia.

BACKGROUND AND METHODS: The incidence and risk factors for erythromycin-induced ototoxicity are unknown. We conducted a prospective, nested case-control study of assessment of auditory function in patients receiving erythromycin versus other antibiotics (control group) for community-acquired pneumonia. Sequential audiograms were performed during antibiotic therapy for both cases and controls by an audiologist unaware of the identity of the therapy administered. Erythromycin serum concentrations were obtained for all patients receiving erythromycin. RESULTS: Symptomatic ototoxicity (tinnitus or hearing loss) confirmed by audiograms was documented in five of 30 patients receiving erythromycin and none of 15 receiving other antibiotics. Ototoxicity was significantly related to high peak concentration and high AUC 0-infinity as a function of decreased total systemic clearance. Ototoxicity occurred only in those patients who received 4 g/day versus 2 g/day or no erythromycin (p = 0.05). Ototoxicity resolved in all patients within 6 to 14 days after discontinuation of therapy. CONCLUSIONS: Erythromycin ototoxicity is dose- and serum concentration-dependent. Patients receiving erythromycin, especially at a total daily dose of 4 g, should be monitored regularly for subjective evidence of sensorineural hearing dysfunction. Ototoxicity is reversible if the diagnosis is made early in the course.

Evaluation of housestaff physicians' preparation and interpretation of sputum Gram stains for community-acquired pneumonia.

OBJECTIVE: To evaluate the preparation and interpretation of sputum Gram stains by housestaff physicians in the assessment of patients with community-acquired pneumonia. DESIGN: A prospective, multicenter study. SETTING: Two university-affiliated hospitals in Pittsburgh. PATIENTS: Ninety-nine cases of clinically and radiographically established pneumonia occurring in 97 patients. Diagnostic test assessment: Housestaff and microbiology personnel prepared a Gram stain for each case of pneumonia. Housestaff assessed the presence and identity of a predominant microbial organism on the slides they prepared. Two senior staff microbiologists, blinded to patient and preparer, evaluated all slides for preparation, sputum purulence, and identification of the predominant organism. Two reference standards were used to assess the sensitivity, specificity, and predictive values of housestaff's Gram-stain interpretations: 1) senior staff microbiologists' determinations of the microbes present using the slides without benefit of culture results, and 2) the etiologic agent derived from results of sputum culture, blood culture, or serology. MEASUREMENTS AND MAIN RESULTS: Housestaff physicians completed a Gram stain in 58% of the pneumonia episodes. Gram stains were not made in 42% of cases, primarily because patients were unable to produce sputum. Fifteen percent of housestaff's smears were judged inadequately prepared, compared with 3% for the laboratory personnel (p less than 0.01). Housestaff obtained purulent sputum samples significantly more often than did nursing personnel (58% versus 38%; p less than 0.01). Housestaff's Gram stains were 90% sensitive for detecting pneumococcus, with a 50% false-positive rate. The sensitivity of the Gram stain was less for identification of Haemophilus influenzae than for identification of Streptococcus pneumoniae. A single antimicrobial agent was chosen as initial therapy for 50% of the patients in whom housestaff identified a predominant organism, compared with 30% in whom a predominant organism was not identified (p less than or equal to 0.05). CONCLUSIONS: Although housestaff obtained purulent sputum samples more frequently than did nursing personnel, they made systematic errors in the preparation and interpretation of Gram-stained slides. Housestaff physicians should receive formal training in the preparation and interpretation of Gram stains; the specific defects elucidated in this study warrant special attention.

Interspecies comparison of renal cortical receptors for parathyroid hormone and parathyroid hormone-related protein.

Parathyroid hormone (PTH) and PTH-related proteins (PTHrP) interact with a common receptor in rat bone cells and in canine renal membranes with similar affinity, but PTHrP are substantially less potent than PTH in stimulating adenylate cyclase in canine renal membranes; in contrast, PTH and PTHrP are equipotent in stimulating adenylate cyclase in rat bone cells. This discrepancy has been largely viewed as reflecting differences in the relative efficiency of signal transduction of PTHrP between bone and kidney assay systems. To test the alternative (but not mutually exclusive) hypothesis that these differences could reflect interspecies differences in PTH receptors, we have characterized the bioactivity of amino-terminal PTHrP and PTH in rat and human renal cortical membranes (RCM) and compared them to results we previously reported in canine RCM. The stability of PTH and PTHrP peptides under binding and adenylate cyclase assay conditions was greater than 80% for each species. Competitive inhibition of [125I](Tyr36)hPTHrP-(1-36)NH2 binding to rat RCM by bPTH-(1-34) and (Tyr36)hPTHrP-(1-36)NH2 yielded nearly identical binding dissociation constants (3.7 and 3.6 nM, respectively), and binding to human RCM demonstrated slightly greater potency for PTHrP (0.5 nM) than for PTH (0.9 nM). Similarly, adenylate cyclase stimulating activity was equivalent for the two peptides in rat RCM, but PTHrP was twofold more potent than PTH in human RCM. Covalent photoaffinity labeling of protease-protected rat RCM yielded an apparent 80 kD receptor protein, and cross-linking of human RCM labeled an 85 kD receptor, indistinguishable in size from the canine renal PTH receptor. We conclude that rat, canine, and human renal cortical PTH receptors exhibit species specificity. The previously observed differences between rat bone cells and canine renal membranes in the efficiency of signal transduction by PTHrP may be explained, at least in part, by these species differences.