Roberta L. McKee

Maintenance of Adult-rat Liver Slices in Dynamic Organ-culture

SummaryAdult rat liver slices were maintained for 20 h in a novel organ culture system with minimal loss of viability and function. Potassium and adenosine triphosphate levels were maintained at in vivo levels, following an initial recovery period (2 to 4 h), for up to 20 h. Protein synthesis and secretion were linear for 20 and 16 h, respectively. In addition, the liver slices synthesized glycogen between 4 and 12 h in culture. Finally, the liver slices were hormonally responsive during the 20 h culture period as exemplified by glucagon-stimulated glucose production. This system provides a simple and effective method for the culture and biochemical maintenance of adult rat liver for 20 h with minimal loss of biochemical function.

Biological activity of parathyroid hormone antagonists substituted at position 13

Lysine occupies position 13 in the parathyroid hormone (PTH) antagonist, [Nle8,18,Tyr34]bPTH(7-34)NH2. Acylation of the epsilon-amino group in lysine 13 by a hydrophobic moiety is well tolerated in terms of bioactivity: the analog [Nle8,18, D-Trp12,Lys 13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(7-34)NH2 is equivalent to the parent peptide in its affinity for PTH receptors and its ability to inhibit PTH-stimulated adenylate cyclase in both kidney- and bone-based assays. Truncation of this peptide by deletion of phenylalanyl7 with concomitant removal of the amino-terminal alpha-amino group yielded the analog desamino[Nle8,18,D-Trp12,Lys13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(8-34)NH2, an antagonist of high potency in vitro (Kb = 4 and 9 nM, Ki = 73 and 3.5 nM in kidney- and bone-based assays, respectively). Also this analog is potentially stable to aminopeptidases present in many biological systems.

Perifused precision-cut liver slice system for the study of hormone-regulated hepatic glucose metabolism.

A nonrecirculatory perfusion system for precision-cut rat liver slices has been developed and utilized for investigating hormone-regulated hepatic glucose metabolism. In this system, slices are cultured in a highly controlled environment and exhibit excellent retention of viability as judged by their maintenance of intracellular potassium and glycogen contents. Using this system, the complex physiological phenomenon of hormone-regulated glycogenolysis was investigated at both extra- and intracellular sites. Specifically, the sensitive responses of intracellular cyclic AMP (cAMP) production, activation of cyclic AMP-dependent protein kinase, and production of glucose upon glucagon stimulation have been measured. The maximal responses observed for these parameters were either equal to or greater than those previously reported for either isolated hepatocytes or perfused livers, demonstrating the sensitivity of this technique. Upon dose-response examination of glucagon challenge, it was observed that high doses of glucagon (greater than 16 nM) stimulate glucose production by activating the cAMP-second messenger cascade. In contrast, low doses (less than 4 nM) stimulate this process without production of intracellular cAMP or activation of cAMP-dependent protein kinase, suggesting the operation of cAMP-independent messenger. Since this system permits measurements of parameters common to many cellular processes, this methodology is suitable for addressing both pharmacological and toxicological questions.

New Directions for the Design of Parathyroid Hormone Antagonists

Peptide hormone antagonists that are effective in vivo are uniquely precise tools for biomedical research. They can be used to determine how peptide hormones act, what their role is in normal physiological processes, and how they contribute to pathophysiologic states.

A tumor-secreted protein associated with human hypercalcemia of malignancy. Biology and molecular biology.

This investigation addresses a theoretical concept of tumor pathogenesis proposed over 40 years ago, namely that malignancy-associated hypercalcemia can result from endocrine secretion by tumors of a PTH-like factor. These studies demonstrate that a fragment of hHCF alone, without added or tumor-secreted cofactors or hormones, can produce hypercalcemia and other biochemical abnormalities associated with HHM. The hypercalcemia can be generated by hHCF-(1-34)NH2 action on bone, although kidney and gut could contribute to the HHM syndrome when it occurs naturally. No other tumor-secreted peptide displays this biological profile. These studies establish one (PTH-like) mechanism by which human tumors could produce hypercalcemia. Furthermore, the finding that hHCF-(1-34)NH2 is more potent than PTH in some systems is of considerable interest for the future design of hormone analogs. A broad spectrum of biological properties of hHCF-(1-34)NH2, including production of components of the HHM syndrome, can be inhibited by a PTH antagonist. Because [Tyr-34]bPTH-(7-34)NH2 selectively and competitively occupies PTH receptors, our studies demonstrate formally that hHCF-(1-34)NH2 mediates some (and perhaps all) of its actions via receptors conventionally regarded as intended for interaction with PTH, but which actually may be present to allow for expression of bioactivity of both secreted proteins. Although some structural homology is shared by the two hormones and many contribute to interaction with receptors, the disparity in structure, especially within the 1-34 domains responsible for bioactivity in both hormones, is more pronounced. The similarity in biological profiles despite structural differences between hHCF and PTH is emphasized by the inhibitory action of [Tyr-34]bPTH-(7-34)NH2 against the tumor peptide even in the absence of much of the homologous region in the PTH antagonist. This investigation provides impetus for designing more potent antagonists, which must now be regarded more appropriately as inhibitors of both PTH and hHCF. Such antagonists may best be generated from hybrid structures of the two hormones. In any case, these studies establish a promising new approach to therapy of tumor-associated hypercalcemia.