Extracellular ATP and some of its analogs induce transient rises in cytosolic free calcium in individual canine keratinocytes.

Changes in intracellular free calcium ([Ca++]i) play an important role in a variety of biochemical reactions that lead to cellular responses such as proliferation and differentiation. The response of [Ca++]i to extracellular nucleotides (ATP, UTP, ITP, and AMP-PNP) was determined in individual canine keratinocytes using the fluorescent probe fura-2 and digital video fluorescence imaging microscopy. In the presence of 1.8 mM extracellular Ca++, 100 and 500 microM ATP caused a rapid (less than 9 sec) three- to twelvefold rise in [Ca++]i above resting levels of 50-150 nM followed by occasional fluctuations. Small responses were elicited with doses as low as 0.1 microM ATP. The response of cells stimulated with 500 microM ATP in Ca(++)-free medium was characterized by 1.5 to 3 times rapid initial peak followed by a decrease of [Ca++]i below resting levels. Loss of response occurred in the majority of keratinocytes preincubated for 30 min in Ca(++)-free medium. UTP was as effective as ATP in stimulating rises in [Ca++]i in keratinocytes. Smaller elevations in [Ca++]i up to four- to fivefold resting levels were noted with 100 microM AMP-PNP or 500 microM ITP. Desensitization of cells was demonstrated when a second stimulation followed the primary ATP or UTP treatment. These results are suggestive of the presence of purinergic receptors in the cytoplasmic membrane of canine keratinocytes. Experiments using the calcium channel blocker lanthanum suggest that ATP-induced initial rises and sustained levels of [Ca++]i are dependent on the release of Ca++ from intracellular stores. These intracellular Ca++ stores appear to be rapidly depleted after removal of extracellular calcium ([Ca++]e), thereby abolishing ATP-induced [Ca++]i increases.

Primary vertebral hemangiosarcoma in a dog.

A 7-year-old spayed Cocker Spaniel was evaluated for recurrent hind limb paraplegia. Radiography and myelography revealed a compression fracture of T-9 secondary to a lytic process. Findings at necropsy and histologic and immunohistochemical examinations revealed 3 sites of hemangiosarcoma in each of the bodies of T-9 to T-11. Primary vertebral hemangiosarcoma is rare, and in this dog may have represented a multicentric occurrence or a primary neoplasm with adjacent metastatic foci.

Calcium-independent increases in pericellular plasminogen activator activity in pemphigus vulgaris.

Intracellular free calcium ([Ca2+]i), an important second messenger, plays a crucial role in a variety of biochemical reactions leading to cell activation and protein secretion. This study examines the potential role of [Ca2+]i in mediating increases in pericellular plasminogen activator activity of canine keratinocytes observed upon binding of human pemphigus vulgaris IgG (hPV IgG). Using the calcium-sensitive fluorescent probe fura-2 and digital video fluorescence imaging microscopy, [Ca2+]i levels were determined in individual keratinocytes for up to 29 minutes after addition of 0.1-5 mg/ml hPV IgG to monolayers of subconfluent and confluent cultures. Extracellular ATP (a known [Ca2+]i-agonist in canine keratinocytes) and normal human IgG (nh IgG) served as positive and negative controls, respectively. HPV IgG and nh IgG failed to induce significant increases in [Ca2+]i, whereas 500 microM ATP induced a rapid, 3- to 12-fold transient increase above resting levels. Binding of hPV IgG to these keratinocyte cultures was demonstrated by immunofluorescence at the end of selected experiments. ATP stimulation of cultures previously treated with hPV IgG showed normal responsiveness and more than 90% of the cells were still viable at the end of [Ca2+]i imaging, thus demonstrating that failure to respond to hPV IgG was not due to an experimental artifact. Plasminogen activator activity in supernatants of confluent cultures incubated with 0.1-1 mg/ml hPV IgG or nh IgG and harvested at various time intervals was dependent on the IgG dose used and increased steadily over time. Increases in activity were 47-92% higher in cultures treated with hPV IgG than those incubated with the same dose of nh IgG.(ABSTRACT TRUNCATED AT 250 WORDS)

Two rare cutaneous neoplasms in horses: apocrine gland adenocarcinoma and carcinosarcoma.

Two rare equine cutaneous neoplasms, an apocrine gland adenocarcinoma and a carcinosarcoma were diagnosed in a 17-year-old pony and a 14-year-old mare, respectively. The apocrine gland adenocarcinoma was present on the prepuce. Histologically, papillary projections of low cuboidal to columnar epithelial cells were generally well differentiated, and surrounded dilated acini. Stromal invasion was present, but vascular and lymphatic invasion was not seen. The carcinosarcoma was present in the right flank of the mare. Two discrete cell populations were characterized histologically. One portion of the mass was composed of elongated, loosely arranged mesenchymatous cells; the second population consisted of dense sheets of pleomorphic, basophilic cells forming irregular acini.

Serum-free conditions for the long term growth and differentiation of neoplastic canine keratinocytes.

Long term cultures of canine keratinocytes have been established but culture conditions currently used require supplementation with fetal bovine serum (FBS). Unfortunately, FBS contains many non-defined components which may interfere with in vitro studies. This study describes the development of defined serum-free culture conditions for neoplastic canine keratinocytes grown submerged and at the air-liquid interface. Two commercially available serum-free media established for human epidermal cells failed to support canine keratinocyte growth. In contrast, a defined serum-free medium developed in our laboratory successfully supported proliferation of neoplastic canine keratinocytes for at least 40 passages. Cells showed a slower growth rate, but reached similar final densities and were morphologically identical to those cultured in FBS. Grown at the air-liquid interface, the cells reached the same degree of differentiation as in vivo stratified squamous epithelium and cultures grown in FBS. These results demonstrate that canine keratinocytes require different serum-free growth conditions than human cells. Neoplastic canine keratinocyte cultures, grown under serum-free culture conditions, provide an ideal in vitro system for comparative studies of keratinocyte biology and pathogenesis of various dermatoses.